Methods of using antibodies against human IL-22

ABSTRACT

The present application provides human antibodies and antigen binding fragments thereof that specifically bind to the human interleukin-22 (IL-22) and methods of using those antibodies, for example, in diagnosing, treating or preventing inflammatory disorders, autoimmune diseases, allergies, septic shock, infectious disorders, transplant rejection, cancer, and other immune system disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. provisionalapplication No. 60/774,595, filed Feb. 21, 2006, the entire disclosureof which is relied upon and incorporated by reference.

TECHNICAL FIELD

This invention relates to antibodies, e.g., human antibodies, andantigen-binding fragments thereof that bind interleukin-22 (IL-22), inparticular, human IL-22, and their use in regulating IL-22-associatedactivities. The antibodies disclosed herein are useful in diagnosing,preventing, and/or treating IL-22 associated disorders, e.g., autoimmunedisorders, including arthritis.

BACKGROUND OF THE INVENTION

Antigens initiate immune responses and activate the two largestpopulations of lymphocytes: T cells and B cells. After encounteringantigen, T cells proliferate and differentiate into effector cells,while B cells proliferate and differentiate into antibody-secretingplasma cells. These effector cells secrete and/or respond to cytokines,which are small proteins (<about 30 kDa) secreted by lymphocytes andother cell types.

Interleukin-22 (IL-22) is a class II cytokine that shows sequencehomology to IL-10. Its expression is up-regulated in T cells by IL-9 orConA (Dumoutier L. et al. (2000) Proc Nat Acad Sci USA 97(18):10144-9).Further studies have shown that expression of IL-22 mRNA is induced invivo in response to LPS administration, and that IL-22 modulatesparameters indicative of an acute phase response (Dumoutier L. et al.(2000); Pittman D. et al. (2001) Genes and Immunity 2:172). In addition,IL-22 enhances the expression of antimicrobial peptides associated withhost defense, including β-defensin, S100A7, S100A8, and S100A. Wolk etal., Immunity, 21:241-54 (2004); Boniface et al., J. Immunol.174:3695-3702 (2005); Liang et al., J. Exp. Med., 203(10):2271-79(2006). Taken together, these observations indicate that IL-22 plays arole in inflammation (Kotenko S. V. (2002) Cytokine & Growth FactorReviews 13(3):223-40).

IL-22 is believed to bind to a receptor complex consisting of IL-22R andIL-10R2, two members of the type II cytokine receptor family (CRF2) (XieM. H. et al. (2000) J Biol Chem 275(40):31335-9; Kotenko S. V. et al.(2001) J Biol Chem 276(4):2725-32). Both chains of the IL-22 receptorare expressed constitutively in a number of organs. Epithelial celllines derived form these organs are responsive to IL-22 in vitro(Kotenko S. V. (2002) Cytokine & Growth Factor Reviews 13(3):223-40).IL-22 induces activation of the JAK/STAT3 and ERK pathways, as well asintermediates of other MAPK pathways (Dumoutier L. et al. (2000) supra;Xie M. H. et al. (2000) supra; Dumoutier L. et al. (2000) J Immunol164(4):1814-9; Kotenko S. V. et al. (2001) J Biol Chem 276(4):2725-32;Lejeune, D. et al. (2002) J Biol Chem 277(37):33676-82).

CRF2 members are receptors for IFNα/β, IFNγ, coagulation factor VIIa,IL-10 and the IL-10 related proteins IL-19, IL-20, IL-22, IL-24, IL-26,as well as the recently identified IFN-like cytokines, IL-28 and IL-29(Kotenko S. V. (2002) Cytokine & Growth Factor Reviews 13(3):223-40;Kotenko, S. V. et al. (2000) Oncogene 19(21):2557-65; Sheppard, P. etal. (2003) Nature Immunology 4(1):63-8; Kotenko, S. V. et al. (2003)Nature Immunology 4(1):69-77). In addition to these membrane receptors,the CRF2 family also includes a soluble protein, IL-22 binding protein(IL-22BP), which is specific for IL-22 and blocks its activity(Dumoutier, L. et al. (2001) J Immunol 166(12):7090-5; Kotenko, S. V. etal. (2001) J Immunol 166(12):7096-103; Xu, W. et al. (2001) Proc NatlAcad Sci USA 98(17):9511-6; Gruenberg, B. H. et al. (2001) Genes &Immunity 2(6):329-34; Wei C-C et al. (2003) Genes & Immunity 4:204-211).While the IL-22 receptor complex is unique for IL-22, each chain (i.e.,IL-22R and IL-10R2) is shared with other CRF2 members to definefunctional receptors for other cytokines, including IL-20, IL-24(IL-22R/IL-20R2), IL-28, IL-29 (IFN-λR1/IL-10R2) and IL-10(IL-10R1/IL-10R2) (Dumoutier, L. et al. (2001) J. Immunol.167(7):3545-9; Wang, M. et al. (2002) J Biol Chem 277(9):7341-7;Parrish-Novak, J. et al. (2002) J Biol Chem 277(49):47517-23; Kotenko,S. V. et al. (1997) EMBO J. 16(19):5894-903; Spencer, S. D. et al.(1998) J Exp Med 187(4):571-8).

Both chains of the CRF2-composed receptor are necessary for signaltransduction. One chain of the composed receptor has been historicallydefined as a ligand binding chain (e.g., IFNγR1) based on its highaffinity for the cytokine. The other chain (e.g., IFNγR2) has beencharacterized as a helper or accessory chain, and shows minimal affinityfor the cytokine alone (Kotenko, S. V. et al. (2000) Oncogene19(21):2557-65). For IL-22, IL-22R is the high affinity receptor subunitwith IL-10R2 subsequently binding to the IL-22/IL-22R complex (Li, J. etal. (2004) Int. Immunopharmacol. 4(5):673-708; Logsdon, N. J. et al.(2002) J. Interferon Cytokine Res 22(11):1099-1112).

SUMMARY OF THE INVENTION

The present application provides, at least in part, IL-22 binding agentssuch as antibodies and antigen-binding fragments thereof that bind tointerleukin-22 (“IL-22”), in particular, human IL-22, with high affinityand specificity. The antibodies and antigen-binding fragments thereof ofthe present invention are also referred to herein as “anti-IL-22antibodies” and “fragments thereof,” respectively. In one embodiment,the antibody or fragment thereof reduces, inhibits, or antagonizes IL-22activity. Such antibodies can be used to regulate immune responses orIL-22-associated disorders by antagonizing IL-22 activity. In otherembodiments, the anti-IL-22 antibody can be used diagnostically, or as atargeting antibody to deliver a therapeutic or a cytotoxic agent to anIL-22-expressing cell. Thus, the anti-IL-22 antibodies of the inventionare useful in diagnosing, treating, and/or preventing IL-22-associateddisorders, e.g., autoimmune disorders, e.g., arthritis (includingrheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis,psoriatic arthritis, lupus-associated arthritis or ankylosingspondylitis), scleroderma, systemic lupus erythematosis, HIV, Sjogren'ssyndrome, vasculitis, multiple sclerosis, autoimmune thyroiditis,dermatitis (including atopic dermatitis and eczematous dermatitis),myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease,colitis, diabetes mellitus (type I); inflammatory conditions of, e.g.,the skin (e.g., psoriasis), cardiovascular system (e.g.,atherosclerosis), nervous system (e.g., Alzheimer's disease), liver(e.g., hepatitis), kidney (e.g., nephritis) and pancreas (e.g.,pancreatitis); cardiovascular disorders, e.g., cholesterol metabolicdisorders, oxygen free radical injury, ischemia; disorders associatedwith wound healing; respiratory disorders, e.g., asthma and COPD (e.g.,cystic fibrosis); acute inflammatory conditions (e.g., endotoxemia,sepsis and septicaemia, toxic shock syndrome and infectious disease);transplant rejection and allergy. In one embodiment, theIL-22-associated disorder is, an arthritic disorder, e.g., a disorderchosen from one or more of rheumatoid arthritis, juvenile rheumatoidarthritis, osteoarthritis, psoriatic arthritis, or ankylosingspondylitis; a respiratory disorder (e.g., asthma, chronic obstructivepulmonary disease (COPD); or an inflammatory condition of, e.g., theskin (e.g., psoriasis), cardiovascular system (e.g., atherosclerosis),nervous system (e.g., Alzheimer's disease), liver (e.g., hepatitis),kidney (e.g., nephritis), pancreas (e.g., pancreatitis), andgastrointestinal organs, e.g., colitis, Crohn's disease and IBD.

Accordingly, in one aspect, the invention features an isolated antibodythat binds to IL-22, in particular, human IL-22. In certain embodiments,the anti-IL-22 antibody may have at least one of the followingcharacteristics: (1) it is a monoclonal or single specificity antibody;(2) it is a human antibody; (3) it is an in vitro generated antibody;(4) it is an in vivo generated antibody (e.g., transgenic system); (5)it binds to IL-22 with an association constant of at least 10¹² M⁻¹; (6)it binds to IL-22 with an association constant of at least 10¹¹ M⁻¹; (7)it binds to IL-22 with an association constant of at least 10¹⁰ M⁻¹; (8)it binds to IL-22 with an association constant of at least 10⁹ M⁻¹; (9)it binds to IL-22 with an association constant of at least 10⁶ M⁻¹; (10)it binds to IL-22 with a dissociation constant of 500 nM or less; (11)it binds to IL-22 with a dissociation constant of 10 nM or less; (12) itbinds to IL-22 with a dissociation constant of 150 pM or less; (13) itbinds to IL-22 with a dissociation constant of 60 pM or less; (14) itinhibits binding of IL-22 to IL-22R or a receptor complex of IL-22R andIL-10R2 with an IC₅₀ of 10 nM or less; (15) it blocks IL-22 mediatedproliferation of IL-22 receptor engineered BaF3 cells with an IC₅₀ of 1nM or less in one embodiment, with an IC₅₀ of 150 pM or less in anotherembodiment, with an IC₅₀ of 100 pM or less in another embodiment, andwith an IC₅₀ of 10 pM or less in another embodiment; and (16) it blocksIL-22 mediated GROa secretion from HT29 cells with an IC₅₀ of 1 nM orless in one embodiment, with an IC₅₀ of 150 pM or less in anotherembodiment, and with an IC₅₀ of 10 pM or less in another embodiment.IL-22 mediated BaF3 cell proliferation and IL-22 mediated GROa secretionfrom HT29 cells can be measured as described in the examples.

Nonlimiting illustrative embodiments of the antibodies of the inventionare referred to herein as “GIL01,” “GIL16,” “GIL45,” “GIL60,” “GIL68,”“GIL92,” “097D09,” “062A09,” “062G05,” “087B03,” “367D04,” “368D04,”“166B06,” “166G05,” “375G06,” “376B10,” “354A08,” “355B06,” “355E04,”and “356A11.” These antibodies can be germlined or non-germlined. Inanother embodiment, the antibody is chosen from 356A11, 354A08, 087B03,and 368D04. The antibodies of the invention may specifically bind to thesame IL-22 epitope or a similar epitope (e.g., an overlapping epitope)that GIL01, GIL16, GIL45, GIL60, GIL68, GIL92, 097D09, 062A09, 062G05,087B03, 367D04, 368D04, 166B06, 166G05, 375G06, 376B10, 354A08, 355B06,355E04, or 356A11 binds to. In other embodiments, the antibodiesspecifically bind to a fragment of an IL-22, e.g., a fragment of atleast 10, 20, 50, 75, 100, 150, or 200 amino acids contiguous to theamino acid sequence set forth in SEQ ID NO:1, or a sequence that is atleast 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical thereto. Inother embodiments, the antibody competitively inhibits the binding of atleast one of GIL01, GIL16, GIL45, GIL60, GIL68, GIL92, 097D09, 062A09,062G05, 087B03, 367D04, 368D04, 166B06, 166G05, 375G06, 376B10, 354A08,355B06, 355E04, or 356A11 to its target epitope.

In one embodiment, the antibody of the present invention includes aV_(H) domain, V_(L) domain, or combination thereof, of the F_(v)fragment of GIL01, GILL 6, GIL45, GIL60, GIL68, GIL92, 097D09, 062A09,062G05, 087B03, 367D04, 368D04, 166B06, 166G05, 375G06, 376B10, 354A08,355B06, 355E04, or 356A11. For example, the antibody includes a V_(H)and/or a V_(L) domain having amino acid sequence as set forth in Tables1 and 7 (SEQ ID NO:5, 23, 41, 59, 77, 95, 113, 131, 149, 167, 185, 203,221, 239, 257, 275, 293, 311, 329, 347, 365, 383, 401, 419, 437, 455,473, 491, 509, 527, 545, 563, 581, 599, or 617 for V_(H) and SEQ IDNO:6, 24, 42, 60, 78, 96, 114, 132, 150, 168, 186, 204, 222, 240, 258,276, 294, 312, 330, 348, 366, 384, 402, 420, 438, 456, 474, 492, 510,528, 546, 564, 582, 600, or 618 for V_(L)), or a sequence substantiallyidentical thereto (e.g., a sequence at least about 85%, 90%, 95%, 96%,97%, 98%, 99% or more identical thereto, or which differs by no morethan 1, 2, 5, 10 or 15 amino acid residues from SEQ ID NO:5, 6, 23, 24,41, 42, 59, 60, 77, 78, 95, 96, 113, 114, 131, 132, 149, 150, 167, 168,185, 186, 203, 204, 221, 222, 239, 240, 257, 258, 275, 276, 293, 294,311, 312, 329, 330, 347, 348, 365, 366, 383, 384, 401, 402, 419, 420,437, 438, 455, 456, 473, 474, 491, 492, 509, 510, 527, 528, 545, 546,563, 564, 581, 582, 599, 600, 617, or 618).

In another embodiment, the antibody of the present invention includes aV_(H) domain, V_(L) domain, or combination thereof, of the F_(v)fragment of an antibody chosen from 356A11, 354A08, 087B03, and 368D04.In this embodiment, the antibody, or antigen-binding fragment thereof,comprises:

a V_(H) domain comprising the amino acid sequence set out in SEQ IDNO:167 or 491 and/or a V_(L) domain comprising the amino acid sequenceset out in SEQ ID NO:168 or 492 (087B03);

a V_(H) domain comprising the amino acid sequence set out in SEQ IDNO:293 or 545 and/or a V_(L) domain having the amino acid sequence setout in SEQ ID NO:294 or 546 (354A08);

a V_(H) domain comprising the amino acid sequence set out in SEQ IDNO:203 or 617 and/or a V_(L) domain comprising the amino acid sequenceset out in SEQ ID NO:204 or 618 (368D04); or

a V_(H) domain comprising the amino acid sequence set out in SEQ IDNO:347 or 599 and/or a V_(L) domain comprising the amino acid sequenceset out in SEQ ID NO:348 or 600 (356A11).

In another embodiment, the antibody includes a V_(H) and/or V_(L) domainencoded by a nucleic acid having a nucleotide sequence as set forth inTables 1 and 7 (SEQ ID NO:14, 32, 50, 68, 86, 104, 122, 140, 158, 176,194, 212, 230, 248, 266, 284, 302, 320, 338, 356, 374, 392, 410, 428,446, 464, 482, 500, 518, 536, 554, 572, 590, 608, or 626 for V_(H) andSEQ ID NO:15, 33, 51, 69, 87, 105, 123, 141, 159, 177, 195, 213, 231,249, 267, 285, 303, 321, 339, 357, 375, 393, 411, 429, 447, 465, 483,501, 519, 537, 555, 573, 591, 609, or 627 for V_(L)), or a sequencesubstantially identical thereto (e.g., a sequence at least about 85%,90%, 95%, 96%, 97%, 98%, 99% or more identical thereto, or which differsby no more than 1, 2, 3, 6, 15, 30 or 45 nucleotides from SEQ ID NO: 14,15, 32, 33, 50, 51, 68, 69, 86, 87, 104, 105, 122, 123, 140, 141, 158,159, 176, 177, 194, 195, 212, 213, 230, 231, 248, 249, 266, 267, 284,285, 302, 303, 320, 321, 338, 339, 356, 357, 374, 375, 392, 393, 410,411, 428, 429, 446, 447, 464, 465, 482, 483, 500, 501, 518, 519, 536,537, 554, 555, 572, 573, 590, 591, 608, 609, 626, or 627).

In other embodiments, the antibody includes an F_(v) domain having anamino acid sequence as set forth in Tables 1 and 7 (SEQ ID NO:7, 25, 43,61, 79, 97, 115, 133, 151, 169, 187, 205, 223, 241, 259, 277, 295, 313,331, 349, 367, 385, 403, 421, 439, 457, 475, 493, 511, 529, 547, 565,583, 601, or 619), Or a sequence substantially identical thereto (e.g.,a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or moreidentical thereto, or which differs by no more than 1, 2, 5, 10, 15, 20,30 or 35 amino acid residues from SEQ ID NO:7, 25, 43, 61, 79, 97, 115,133, 151, 169, 187, 205, 223, 241, 259, 277, 295, 313, 331, 349, 367,385, 403, 421, 439, 457, 475, 493, 511, 529, 547, 565, 583, 601, or619). In another embodiment, the antibody of the present inventionincludes an F_(v) domain of an antibody chosen from 356A11 (SEQ IDNO:349 or 601), 354A08 (SEQ ID NO:295 or 547), 087B03 (SEQ ID NO:169 or493), and 368D04 (SEQ ID NO:205 or 619). In another embodiment, theantibody includes an F_(v) domain encoded by a nucleic acid having anucleotide sequence as set forth in Tables 1 and 7 (SEQ ID NO:16, 34,52, 70, 88, 106, 124, 142, 160, 178, 196, 214, 232, 250, 268, 286, 304,322, 340, 358, 376, 394, 412, 430, 448, 466, 484, 502, 520, 538, 556,574, 592, 610, or 628), or a sequence substantially identical thereto(e.g., a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% ormore identical thereto, or which differs by no more than 1, 2, 3, 6, 15,30, 45, 60, 90 or 105 nucleotides from SEQ ID NO: 16, 34, 52, 70, 88,106, 124, 142, 160, 178, 196, 214, 232, 250, 268, 286, 304, 322, 340,358, 376, 394, 412, 430, 448, 466, 484, 502, 520, 538, 556, 574, 592,610, or 628). In yet other embodiments, the antibody comprises at leastone complementarity determining region (CDR) of these V_(H) and V_(L)domains. For example, the antibody can include one, two, or three CDR'sof the V_(H) domain having an amino acid sequence as set forth in orincluded within the sequences in Tables 1 and 7 (SEQ ID NO:5, 7, 8, 9,10, 23, 25, 26, 27, 28, 41, 43, 44, 45, 46, 59, 61, 62, 63, 64, 77, 79,80, 81, 82, 95, 97, 98, 99, 100, 113, 115, 116, 117, 118, 131, 133, 134,135, 136, 149, 151, 152, 153, 154, 167, 169, 170, 171, 172, 185, 187,188, 189, 190, 203, 205, 206, 207, 208, 221, 223, 224, 225, 226, 239,241, 242, 243, 244, 257, 259, 260, 261, 262, 275, 277, 278, 279, 280,293, 295, 296, 297, 298, 311, 313, 314, 315, 316, 329, 331, 332, 333,334, 347, 349, 350, 351, 352, 365, 367, 368, 369, 370, 383, 385, 386,387, 388, 401, 403, 404, 405, 406, 419, 421, 422, 423, 424, 437, 439,440, 441, 442, 455, 457, 458, 459, 460, 473, 475, 476, 477, 478, 491,493, 494, 495, 496, 509, 511, 512, 513, 514, 527, 529, 530, 531, 532,545, 547, 548, 549, 550, 563, 565, 566, 567, 568, 581, 583, 584, 585,586, 599, 601, 602, 603, 604, 617, 619, 620, 621, or 622), or a sequencesubstantially homologous thereto (e.g., a sequence at least about 85%,90%, 95%, 96%, 97%, 98%, 99% or more identical thereto). In anotherembodiment, the antibody of the present invention includes one, two, orthree CDR's of the V_(H) domain of an antibody chosen from 356A11,354A08, 087B03, and 368D04. In this embodiment, the antibody, orantigen-binding fragment thereof, comprises a heavy chain variableregion comprising:

a) SEQ ID NO:170 or 494; b) SEQ ID NO: 171 or 495; and/or c) SEQ ID NO:172 or 496 (087B03);

a) SEQ ID NO:296 or 548; b) SEQ ID NO:297 or 549; and/or c) SEQ IDNO:298 or 550 (354A08);

a) SEQ ID NO:206 or 620; b) SEQ ID NO:207 or 621; and/or c) SEQ IDNO:208 or 622 (368D04); or

a) SEQ ID NO:350 or 602; b) SEQ ID NO:351 or 603; and/or c) SEQ IDNO:352 or 604 (356A11).

In another embodiment, the antibody can include one, two, or three CDR'sof the V_(L) domain having an amino acid sequence as set forth in orincluded within the sequences in Tables 1 and 7 (SEQ ID NO:6, 7, 11, 12,13, 24, 25, 29, 30, 31, 42, 43, 47, 48, 49, 60, 61, 65, 66, 67, 78, 79,83, 84, 85, 96, 97, 101, 102, 103, 114, 115, 119, 120, 121, 132, 133,137, 138, 139, 150, 151, 155, 156, 157, 168, 169, 173, 174, 175, 186,187, 191, 192, 193, 204, 205, 209, 210, 211, 222, 223, 227, 228, 229,240, 241, 245, 246, 247, 258, 259, 263, 264, 265, 276, 277, 281, 282,283, 294, 295, 299, 300, 301, 312, 313, 317, 318, 319, 330, 331, 335,336, 337, 348, 349, 353, 354, 355, 366, 367, 371, 372, 373, 384, 385,389, 390, 391, 402, 403, 407, 408, 409, 420, 421, 425, 426, 427, 438,439, 443, 444, 445, 456, 457, 461, 462, 463, 474, 475, 479, 480, 481,492, 493, 497, 498, 499, 510, 511, 515, 516, 517, 528, 529, 533, 534,535, 546, 547, 551, 552, 553, 564, 565, 569, 570, 571, 582, 583, 587,588, 589, 600, 601, 605, 606, 607, 618, 619, 623, 624, or 625), or asequence substantially identical thereto (e.g., a sequence at leastabout 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical thereto). Inanother embodiment, the antibody of the present invention includes one,two, or three CDR's of the V_(L) domain of an antibody chosen from356A11, 354A08, 087B03, and 368D04. In this embodiment, the antibody, orantigen-binding fragment thereof, comprises a light chain variableregion comprising:

a) SEQ ID NO:173 or 497; b) SEQ ID NO: 174 or 498; and/or c) SEQ IDNO:175 or 499 (087B03);

a) SEQ ID NO:299 or 551; b) SEQ ID NO:300 or 552; and/or c) SEQ IDNO:301 or 553 (354A08);

a) SEQ ID NO:209 or 623; b) SEQ ID NO:210 or 624; and/or c) SEQ IDNO:211 or 625 (368D04); or

a) SEQ ID NO:353 or 605; b) SEQ ID NO:354 or 606; and/or c) SEQ IDNO:355 or 607 (356A11).

In a still further embodiment, the antibody comprises an H3 fragment ofthe V_(H) domain of GIL01, GIL16, GIL45, GIL60, GIL68, GIL92, 097D09,062A09, 062G05, 087B03, 367D04, 368D04, 166B06, 166G05, 375G06, 376B10,354A08, 355B06, 355E04, or 356A11, e.g., an H3 fragment having the aminoacid sequence as set forth in Tables 1 and 7 (SEQ ID NO:10, 28, 46, 64,82, 100, 118, 136, 154, 172, 190, 208, 226, 244, 262, 280, 298, 316,334, 352, 370, 388, 406, 424, 442, 460, 478, 496, 514, 532, 550, 568,586, 604, or 622), or a sequence substantially identical thereto (e.g.,a sequence at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or moreidentical thereto).

The antibody of the invention can be full-length (e.g., include at leastone complete heavy chain and at least one complete light chain) or caninclude only an antigen-binding fragment (e.g., a Fab, F(ab′)₂, Fv, asingle chain F_(v) fragment, a Fd fragment, or a dAb fragment). Theantibody can include a constant region, or a portion thereof, chosenfrom any of: the kappa, lambda, alpha, gamma, delta, epsilon and muconstant region genes. For example, heavy chain constant regions of thevarious isotypes can be used, including: IgG₁, IgG₂, IgG₃, IgG₄, IgM,IgA₁, IgA₂, IgD, and IgE. The light chain constant region can be chosenfrom kappa or lambda. The antibody may be an IgG, or it may also beIgG_(1κ) or IgG_(1γ).

The anti-IL-22 antibody described herein can be derivatized or linked toanother functional molecule (such as another peptide or protein (e.g., aFab fragment)). For example, an antibody of the invention can befunctionally linked (e.g., by chemical coupling, genetic fusion,non-covalent association or otherwise) to at least one other molecularentity, such as another antibody (e.g., a bispecific or a multispecificantibody), toxin, radioisotope, cytotoxic or cytostatic agent, amongothers.

In another aspect, the invention features a pharmaceutical compositioncontaining at least one anti-IL-22 antibody and a pharmaceuticallyacceptable carrier. The pharmaceutical composition can further include acombination of at least one anti-IL-22 antibody and at least onetherapeutic agent (e.g., cytokine and growth factor inhibitors,immunosuppressants, anti-inflammatory agents, metabolic inhibitors,enzyme inhibitors, cytotoxic agents, cytostatic agents, or combinationsthereof, as described in more detail herein). Combinations of theanti-IL-22 antibody and a therapeutic agent are also within the scope ofthe invention. The compositions and combinations of the invention can beused to regulate IL-22-associated inflammatory conditions, e.g., bymodulating IL-22 signaling through its receptors located on epithelialcells of a variety of tissues, including, but not limited to, those ofthe pancreas, skin, lung, gut, liver, kidney, salivary gland, andvascular endothelia, in addition to potentially activated and tissuelocalized immune cells.

In another aspect, the invention features a method of treating a subjectwith an IL-22-associated disorder. The method includes administering tothe subject an anti-IL-22 antibody in an amount sufficient to inhibit atleast one IL-22 activity of immune cells, thereby treating theIL-22-associated disorder.

The anti-IL-22 antibody can be administered to the subject, alone or incombination, with other therapeutic agents as described herein. Thesubject may be a mammal, e.g. human. For example, the method can be usedto treat a subject with an IL-22-associated disorder such as autoimmunedisorders, e.g., arthritis (including rheumatoid arthritis, juvenilerheumatoid arthritis, osteoarthritis, psoriatic arthritis,lupus-associated arthritis or ankylosing spondylitis), scleroderma,systemic lupus erythematosis, HIV, Sjogren's syndrome, vasculitis,multiple sclerosis, autoimmune thyroiditis, dermatitis (including atopicdermatitis and eczematous dermatitis), myasthenia gravis, inflammatorybowel disease (IBD), Crohn's disease, colitis, diabetes mellitus (typeI); inflammatory conditions of, e.g., the skin (e.g., psoriasis),cardiovascular system (e.g., atherosclerosis), nervous system (e.g.,Alzheimer's disease), liver (e.g., hepatitis), kidney (e.g., nephritis)and pancreas (e.g., pancreatitis); cardiovascular disorders, e.g.,cholesterol metabolic disorders, oxygen free radical injury, ischemia;disorders associated with wound healing; respiratory disorders, e.g.,asthma and COPD (e.g., cystic fibrosis); acute inflammatory conditions(e.g., endotoxemia, sepsis and septicaemia, toxic shock syndrome andinfectious disease); transplant rejection and allergy. In oneembodiment, the IL-22-associated disorder is, an arthritic disorder,e.g., a disorder chosen from one or more of rheumatoid arthritis,juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, orankylosing spondylitis; a respiratory disorder (e.g., asthma, chronicobstructive pulmonary disease (COPD); or an inflammatory condition of,e.g., the skin (e.g., psoriasis), cardiovascular system (e.g.,atherosclerosis), nervous system (e.g., Alzheimer's disease), liver(e.g., hepatitis), kidney (e.g., nephritis), pancreas (e.g.,pancreatitis), and gastrointestinal organs, e.g., colitis, Crohn'sdisease and IBD.

In another aspect, the invention features a method of decreasing,inhibiting or reducing an acute phase response in a subject. The methodincludes administering to the subject an IL-22 binding agent, e.g., anIL-22 antagonist, (e.g., an anti-IL-22 antibody or fragment thereof asdescribed herein), in an amount sufficient to decrease, inhibit orreduce the acute phase response in the subject. In one embodiment, thesubject is a mammal, e.g., a human suffering from an IL-22-associateddisorder, including, e.g., respiratory disorders, inflammatory disordersand autoimmune disorders. In one embodiment, the IL-22 binding agent isadministered locally, e.g., topically, subcutaneously, or otheradministrations that are not in the general circulation.

In another aspect, an IL-22 binding agent can be used to alter the typeof immune response and/or increase the efficacy of a vaccine formulationused to immunize a subject. For example, an anti-IL-22 antibody of thepresent invention can be administered before, during and/or after animmunization to increase vaccine efficacy. In one embodiment, thevaccine formulation contains one or more IL-22 antagonists and anantigen, i.e., an immunogen, including, for example, viral, bacterial,or tumor antigens. In another embodiment, the IL-22 antagonist and theimmunogen are administered separately, e.g., within one hour, threehours, one day or two days of each other.

In another aspect, the invention provides a method for detecting thepresence of IL-22 in a sample in vitro. Samples may include biologicalsamples such as serum, plasma, tissue and biopsy. The subject method canbe used to diagnose a disorder, such as an IL-22-associated disorder asdescribed herein. The method includes: (1) contacting the sample or acontrol sample with an anti-IL-22 antibody, and (2) detecting formationof a complex between the anti-IL-22 antibody and the sample or thecontrol sample, wherein a statistically significant change in theformation of the complex in the sample relative to a control sample, isindicative of the presence of the IL-22 in the sample.

In another aspect, the invention provides a method for detecting thepresence of IL-22 in vivo (e.g., in vivo imaging in a subject). Themethod can be used to diagnose a disorder, e.g., an IL-22-associateddisorder as described herein. The method includes: (1) administering ananti-IL-22 antibody to a subject or a control subject under conditionsthat allow binding of the antibody to IL-22, and (2) detecting formationof a complex between the antibody and IL-22, wherein a statisticallysignificant change in the formation of the complex in the subjectrelative to a control, e.g., a control subject, is indicative of thepresence of IL-22.

The antibody may be directly or indirectly labeled with a detectablesubstance to facilitate detection of the bound or unbound antibody.Suitable detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials and radioactivematerials.

In another aspect, the invention provides a method for delivering ortargeting an agent, e.g., a therapeutic or a cytotoxic agent, to anIL-22-expressing cell in vivo. The method includes administering ananti-IL-22 antibody to a subject under conditions that allow binding ofthe antibody to IL-22. The antibody may be coupled to a secondtherapeutic moiety, such as a toxin.

The disclosure provides nucleic acid sequences from the V_(H) and V_(L)domains of GIL01, GIL16, GIL45, GIL60, GIL68, GIL92, 097D09, 062A09,062G05, 087B03, 367D04, 368D04, 166B06, 166G05, 375G06, 376B10, 354A08,355B06, 355E04, and 356A11. Also provided are nucleic acid sequencesthat comprise at least one CDR from GIL01, GIL16, GIL45, GIL60, GIL68,GIL92, 097D09, 062A09, 062G05, 087B03, 367D04, 368D04, 166B06, 166G05,375G06, 376B10, 354A08, 355B06, 355E04, and 356A11. The disclosure alsoprovides vectors and host cells comprising such nucleic acids.

The disclosure further provides methods of producing new V_(H) and V_(L)domains and functional antibodies comprising all or a portion of suchdomains derived from the V_(H) or V_(L) domains of GIL01, GIL16, GIL45,GIL60, GIL68, GIL92, 097D09, 062A09, 062G05, 087B03, 367D04, 368D04,166B06, 166G05, 375G06, 376B10, 354A08, 355B06, 355E04, or 356A11.

Additional aspects of the disclosure will be set forth in part in thedescription, and in part will be obvious from the description, or may belearned by practicing the invention. The invention is set forth andparticularly pointed out in the claims, and the disclosure should not beconstrued as limiting the scope of the claims. The following detaileddescription includes exemplary representations of various embodiments ofthe invention, which are not restrictive of the invention as claimed.The accompanying figures constitute a part of this specification and,together with the description, serve only to illustrate embodiments andnot limit the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Potency of parent anti-IL-22 scFv clones in the IL-22 receptorcomplex assay: bio.IL-22 binding IL-22 receptor complex DELFIAcompetition assay.

FIG. 2. Profiling of lead scFv clones in IL-22 receptor complex assay:bio.IL-22 binding IL-22 receptor complex DELFIA competition assay. (A)GIL 1 derived. (B) GIL 16 derived. (C) GIL 16, GIL 60, and GIL 68derived. (D) GIL 60 derived. (E) GIL 68 derived. (F) GIL 68 derived. (G)GIL 92 derived.

FIG. 3. IgG potency in GROa cell based assays. Optimized GIL-IgGs inhuIL-22 GROa assay. (A) Germlined IgG. (B) Non-germlined IgG.

FIG. 4. Cross species reactivity of IL-22 antibodies by ELISA. OptimizedGIL-IgGs specifically bind to IL-22. (A) Germlined IgG. (B)Non-germlined IgG.

FIG. 5. Amino acid and nucleotide sequences of human IL-22. Thenucleotide sequence of human IL-22 is SEQ ID NO:2 and includes a poly(A) tail. The disclosed nucleotide sequence includes an open readingframe and the amino acid sequence of full-length IL-22 proteincorresponding to the foregoing nucleotide sequence is reported in SEQ IDNO:1. The amino acid sequence of mature IL-22 corresponds to about aminoacids 34-179 of SEQ ID NO:1.

FIG. 6. Amino acid and nucleotide sequences of mouse IL-22.

FIG. 7. Amino acid and nucleotide sequences of non-germlined GIL01,GIL16, GIL45, GIL60, GIL68, GIL92, 097D09, 062A09, 062G05, 087B03,367D04, 368D04, 166B06, 166G05, 375G06, 376B10, 354A08, 355B06, 355E04,and 356A11, including V_(H) and V_(L) domains, and CDRs (H1, H2, H3, L1,L2, and L3).

FIG. 8. Amino acid and nucleotide sequences of germlined GIL01, GIL16,GIL45, GIL60, GIL68, GIL92, 062A09, 087B03, 166B06, 166G05, 354A08,355B06, 355E04, 356A11, and 368D04, including V_(H) and V_(L) domains,and CDRs (H1, H2, H3, L1, L2, and L3).

FIG. 9. Amino acid and nucleotide sequences of scFv's for non-germlinedGIL01, GIL16, GIL45, GIL60, GIL68, GIL92, 097D09, 062A09, 062G05,087B03, 367D04, 368D04, 166B06, 166G05, 375G06, 376B10, 354A08, 355B06,355E04, and 356A11, with CDRs underlined (H1, H2, H3, L1, L2, and L3).

FIG. 10. Amino acid and nucleotide sequences of scFv's for germlinedGIL01, GIL16, GIL45, GIL60, GIL68, GIL92, 062A09, 087B03, 166B06,166G05, 354A08, 355B06, 355E04, 356A11, and 368D04 with CDRs underlined(H1, H2, H3, L1, L2, and L3).

FIGS. 11A-B. In vivo half life of (A) 356A11 and (B) 368D04.

FIGS. 12A-B. Mean disease severity scores (A) or disease severity scores(B) with various doses of 356A11 administered every other day in murineCIA model.

FIGS. 13A-D. Mean disease severity scores of 8 mg kg⁻¹ of 356A11administered every other day in murine CIA model in multiple studies.

FIGS. 14A-B. Mean disease severity scores of 8 mg kg⁻¹ of 356A11administered once or twice a week in murine CIA model.

FIGS. 15A-B. Disease severity scores from two separate studies of 8 mgkg⁻¹ of 356A11 administered every other day, twice a week, or once aweek in murine CIA model.

FIGS. 16A-F. Histological evaluation (paw) of disease progression inmultiple studies using mice treated with (A) 8 mg kg⁻¹ of 356A11 everyother day or (B) 16 mg kg⁻¹ of 356A11 every other day, (C) 8 mg kg⁻¹ of356A11 every other day, once a week, or twice a week, (D) 8 mg kg⁻¹ of356A11 once a week, (E) 8 mg kg⁻¹ of 356A11 twice a week, or (F) 8 mgkg⁻¹ of 356A11 every other day in murine CIA model.

FIG. 17. Detection and stabilization of in vivo IL-22 (ng/mL) inarthritic mice treated with 356A11.

FIG. 18. Detection of in vivo IL-22 (ng/mL) in arthritic miceadministered an isotype control antibody.

FIG. 19. Upregulation of IL-22 and IL-22R protein in human psoriaticlesions.

FIG. 20. Disease evaluation of in mice treated with 16 mg/kg of 356A11,368D04, or control antibody in murine model of psoriasis. (A) Clinicaldisease progression over 10 weeks. (B) Clinical score at 10 weeks.

FIG. 21. Detection of in vivo serum levels of IL-22 (pg/mL) in psoriaticmice treated with 16 mg/kg of 356A11, 368D04, or a control antibody.

FIG. 22. Detection of in vivo serum levels of (A) IL-17A, (B) IL-17F,(C) IL-17A/F; and (D) IL-6 in psoriatic mice treated with 16 mg/kg of356A11, 368D04, or a control antibody.

FIG. 23. Flow cytometric analysis of pooled CD4+ lymph node cellstreated with PMA and ionomycin following isolation from psoriatic micetreated with 16 mg/kg of 356A111, 368D04, or a control antibody andstained for IL-22 and IL-17A; IL-22 and IL-17F; IL-17A and IL-17F; IL-22and TNFα; IL-22 and IFNγ; or TNFα and IFNγ.

FIG. 24. Cytokine gene expression in the ears of psoriatic mice treatedwith 16 mg/kg of 356A11, 368D04, or a control antibody. (A) IL-22. (B)IL-17. (C) IFNγ. (D) IL-17F. (E) IL-6.

FIG. 25. Cytokine detection in the supernatants of pooled lymph nodecells isolated from psoriatic mice treated with 16 mg/kg of 356A11,368D04, or a control antibody and stimulated ex vivo with and withoutplatebound, anti-CD3 antibody. (A) IL-22. (B) IL-6. (C) IFNγ (D) IL-17F.(E) IL-17A/F. (F) IL-17A.

FIG. 26. Cytokine gene expression in pooled lymph node cells isolatedfrom psoriatic mice treated with 16 mg/kg of 356A11, 368D04, or acontrol antibody and stimulated ex vivo with platebound, anti-CD3antibody. (A) IL-22. (B) IL-6. (C) IFNγ (D) IL-17F. (E) IL-17A.

FIG. 27. Disease evaluation of in mice treated with 16 mg/kg of 356A11or control antibody in murine model of psoriasis. Mice treated withIL-12 and LPS at day 1 following adoptive transfer of T cells. (A)Clinical disease progression over 10 weeks. (B) Clinical score at 10weeks.

FIG. 28. Detection of in vivo serum levels of IL-22 (ng/mL) in psoriaticmice treated with 16 mg/kg of 356A11 or a control antibody. Mice treatedwith IL-12 and LPS at day 1 following adoptive transfer of T cells.

FIG. 29. Detection of in vivo serum levels of (A) IL-17A, (B) IL-17F,(C) IL-17A/F; and (D) IL-6 in psoriatic mice treated with 16 mg/kg of356A11 or a control antibody. Mice treated with IL-12 and LPS at day 1following adoptive transfer of T cells.

FIG. 30. Cytokine gene expression in the ears of psoriatic mice treatedwith 16 mg/kg of 356A11 or a control antibody. (A) IL-17. (B) IL-22. (C)IL-17F. (D) IFNγ. (E) IL-6. Mice treated with IL-12 and LPS at day 1following adoptive transfer of T cells.

FIG. 31. Flow cytometric analysis of pooled CD4+ lymph node cellstreated with PMA and ionomycin following isolation from psoriatic micetreated with 16 mg/kg of 356A11 or a control antibody and stained forIL-22 and IL-17A; IL-22 and IL-17F; IL-17A and IL-17F; IL-22 and TNFα;IL-22 and IFNγ; or TNFα and IFNγ. Mice treated with IL-12 and LPS at day1 following adoptive transfer of T cells.

FIG. 32. Cytokine detection in the supernatants of pooled lymph nodecells isolated from psoriatic mice treated with 16 mg/kg of 356A11 or acontrol antibody and stimulated ex vivo with or without platebound,anti-CD3 antibody. (A) IL-22. (B) IL-6. (C) IFNγ (D) IL-17A. (E) IL-17F.Mice treated with IL-12 and LPS at day 1 following adoptive transfer ofT cells.

FIG. 33. Cytokine gene expression in pooled lymph node cells isolatedfrom psoriatic mice treated with 16 mg/kg of 356A11 or a controlantibody and stimulated ex vivo with platebound, anti-CD3 antibody. (A)IL-22. (B) IL-6. (C) IFNγ (D) IL-17A. (E) IL-17F. Mice treated withIL-12 and LPS at day 1 following adoptive transfer of T cells.

FIG. 34. Disease evaluation of in mice treated with 16 mg/kg of 356A11or control antibody in murine model of psoriasis. (A) Clinical diseaseprogression over 10 weeks. (B) Clinical score at 10 weeks.

FIG. 35. Detection of in vivo serum levels of IL-22 (ng/mL) in psoriaticmice treated with 16 mg/kg of 356A11 or a control antibody.

FIG. 36. Cytokine gene expression in the ears of psoriatic mice treatedwith 16 mg/kg of 356A11 or a control antibody. (A) IL-22, (B) IL-17, (C)IL-17F, (D) IL-1F6 (IL-1 family member 6), (E) IL-6, (F) IFNγ, (G) IL-22BP (IL-22 binding protein), or (H) IL-22R1 (IL-22 receptor subunit).

FIG. 37. Disease evaluation of in mice treated with 16 mg/kg of 356A11or control antibody in murine model of psoriasis. (A) Clinical diseaseprogression over 10 weeks. (B) Clinical score at 10 weeks. Mice treatedwith IL-12 and LPS at day 1 following adoptive transfer of T cells.

FIG. 38. Detection of in vivo serum levels of IL-22 (ng/mL) in psoriaticmice treated with 16 mg/kg of 356A11 or a control antibody. Mice treatedwith IL-12 and LPS at day 1 following adoptive transfer of T cells.

FIG. 39. Detection of in vivo serum levels of (A) IL-17A and (B) IL-6 inpsoriatic mice treated with 16 mg/kg of 356A11 or a control antibody(without coadministration of IL-12 and LPS) and (C) IL-17A and (D) IL-6in psoriatic mice treated with 16 mg/kg of 356A11 or a control antibody(IL-12 and LPS coadministered at day 1 following adoptive transfer of Tcells).

FIG. 40. (A). Mean disease severity score or (B) disease severity scoreat day 30 of 16 mg kg⁻¹ of 356A11 administered every other day in murineCIA model.

FIG. 41. Mean disease severity score of 8 mg kg⁻¹ of 356A11 administeredevery other day, once a week, or twice a week in murine CIA model.

FIG. 42. Histological evaluation (paw) of disease progression in micetreated with (A) 8 mg kg⁻¹ of 356A11 once a week, (B) 8 mg kg⁻¹ of356A11 twice a week, or (C) 8 mg kg⁻¹ of 356A11 every other day.

FIG. 43. Histological evaluation (mean paw severity score) of diseaseprogression in mice treated with 8 mg kg 1 of 356A11 once a week, twicea week, or every other day.

FIGS. 44A-B. Mean disease severity scores from two separate studies of 8mg kg⁻¹ of 356A11 administered once a week in murine CIA model.

FIGS. 45A-B. Histological evaluation (paw) of disease progression fromtwo separate studies in mice treated with 8 mg kg⁻¹ of 356A11 once aweek in murine CIA model.

FIGS. 46A-C. Mean disease severity scores from three separate studies of8 mg kg⁻¹ of 368D04 administered once a week in murine CIA model.

FIGS. 47A-C. Histological evaluation (paw) of disease progression fromthree separate studies in mice treated with 8 mg kg⁻¹ of 368D04 once aweek in murine CIA model.

FIG. 48. Serum levels of (A) IL-6 (pg/ml) and (B) CXCL1 (ng/ml) frommice treated with 356A11 (8 mpk once a week) in murine CIA model.

DETAILED DESCRIPTION

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “antibody” refers to an immunoglobulin or fragment thereof, andencompasses any polypeptide comprising an antigen-binding fragment or anantigen-binding domain. The term includes but is not limited topolyclonal, monoclonal, monospecific, polyspecific, non-specific,humanized, human, single-chain, chimeric, synthetic, recombinant,hybrid, mutated, grafted, and in vitro generated antibodies. Unlesspreceded by the word “intact”, the term “antibody” includes antibodyfragments such as Fab, F(ab′)₂, Fv, scFv, Fd, dAb, and other antibodyfragments that retain antigen-binding function. Typically, suchfragments would comprise an antigen-binding domain.

The terms “antigen-binding domain” and “antigen-binding fragment” referto a part of an antibody molecule that comprises-amino acids responsiblefor the specific binding between antibody and antigen. The part of theantigen that is specifically recognized and bound by the antibody isreferred to as the “epitope.” An antigen-binding domain may comprise anantibody light chain variable region (V_(L)) and an antibody heavy chainvariable region (V_(H)); however, it does not have to comprise both. Fdfragments, for example, have two V_(H) regions and often retain someantigen-binding function of the intact antigen-binding domain. Examplesof antigen-binding fragments of an antibody include (1) a Fab fragment,a monovalent fragment having the V_(L), V_(H), C_(L) and C_(H)1 domains;(2) a F(ab′)₂ fragment, a bivalent fragment having two Fab fragmentslinked by a disulfide bridge at the hinge region; (3) a Fd fragmenthaving the two V_(H) and C_(H)1 domains; (4) a F_(v) fragment having theV_(L) and V_(H) domains of a single arm of an antibody, (5) a dAbfragment (Ward et al., (1989) Nature 341:544-546), which has a V_(H)domain; (6) an isolated complementarity determining region (CDR); and(7) a single chain F_(v) (scFv). Although the two domains of the F_(v)fragment, V_(L) and V_(H), are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain F_(v)(scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). These antibodyfragments are obtained using conventional techniques known to those withskill in the art, and the fragments are evaluated for function in thesame manner as are intact antibodies.

The term “effective amount” refers to a dosage or amount that issufficient to regulate IL-22 activity to ameliorate clinical symptoms orachieve a desired biological outcome, e.g., decreased T cell and/or Bcell activity, suppression of autoimmunity, suppression of transplantrejection, etc.

The term “human antibody” includes antibodies having variable andconstant regions corresponding substantially to human germlineimmunoglobulin sequences known in the art, including, for example, thosedescribed by Kabat et al. (See Kabat, et al. (1991) Sequences ofProteins of Immunological Interest, Fifth. Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242). The humanantibodies of the invention may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo), for example in the CDRs, and in particular, CDR3. The humanantibody can have at least one, two, three, four, five, or morepositions replaced with an amino acid residue that is not encoded by thehuman germline immunoglobulin sequence.

The phrase “inhibit” or “antagonize” IL-22 activity and its cognatesrefer to a reduction, inhibition, or otherwise diminution of at leastone activity of IL-22 due to binding an anti-IL-22 antibody, wherein thereduction is relative to the activity of IL-22 in the absence of thesame antibody. The activity can be measured using any technique known inthe art, including, for example, as described in Examples 7 and 9.Inhibition or antagonism does not necessarily indicate a totalelimination of the IL-22 polypeptide biological activity. A reduction inactivity may be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, ormore.

The term “interleukin-22” or “IL-22” refers to a class II cytokine(which may be mammalian) capable of binding to IL-22R and/or a receptorcomplex of IL-22R and IL-10R2, and has at least one of the followingfeatures: (1) an amino acid sequence of a naturally occurring mammalianIL-22 polypeptide (full length or mature form) or a fragment thereof,e.g., an amino acid sequence shown as SEQ ID NO:1 (human) or SEQ ID NO:3(murine) or a fragment thereof; (2) an amino acid sequence substantiallyidentical to, e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identicalto, an amino acid sequence shown as SEQ ID NO:1 or amino acids 34-179thereof (human) or SEQ ID NO:3 (murine) or a fragment thereof; (3) anamino acid sequence which is encoded by a naturally occurring mammalianIL-22 nucleotide sequence or a fragment thereof (e.g., SEQ ID NO:2 ornucleotides 71 to 610 (human) or SEQ ID NO:4 (murine) or a fragmentthereof); (4) an amino acid sequence encoded by a nucleotide sequencewhich is substantially identical to, e.g., at least 85%, 90%, 95%, 96%,97%, 98%, 99% identical to, a nucleotide sequence shown as SEQ ID NO:2or nucleotides 71 to 610 thereof (human) or SEQ ID NO:4 (murine) or afragment thereof; (5) an amino acid sequence encoded by a nucleotidesequence degenerate to a naturally occurring IL-22 nucleotide sequenceor a fragment thereof, e.g., SEQ ID NO:2 (human) or SEQ ID NO:4 (murine)or a fragment thereof; or (6) a nucleotide sequence that hybridizes toone of the foregoing nucleotide sequences under stringent conditions,e.g., highly stringent conditions. The IL-22 may bind to IL-22R and/or areceptor complex of IL-22R and IL-10R2 of mammalian origin, e.g., humanor mouse.

The nucleotide sequence and the predicted amino acid sequence of humanIL-22 are shown in SEQ ID NO:2 and SEQ ID NO:1, respectively. The aminoacid sequence of mature human IL-22 corresponds to amino acids 34-179 ofSEQ ID NO:1. Analysis of recombinant human IL-22 reveals many structuraldomains. (Nagem et al. (2002) Structure, 10:1051-62; U.S. PatentApplication No. US 2002/0187512 A1).

The term “IL-22 activity” refers to at least one cellular processinitiated or interrupted as a result of IL-22 binding to a receptorcomplex consisting of IL-22R and IL-10R2 on the cell. IL-22 activitiesinclude at least one of, but are not limited to: (1) binding IL-22R or areceptor complex of IL-22R and IL-10R2 (e.g., human IL-22R with orwithout human IL-10R2); (2) associating with signal transductionmolecules (e.g., JAK-1); (3) stimulating phosphorylation of STATproteins (e.g., STAT5, STAT3, or combination thereof); (4) activatingSTAT proteins; and (5) modulating (e.g., increasing or decreasing)proliferation, differentiation, effector cell function, cytolyticactivity, cytokine secretion, survival, or combinations thereof, ofepithelial cells, fibroblasts, or immune cells. Epithelial cellsinclude, but are not limited to, cells of the skin, gut, liver, andkidney, as well as endothelial cells. Fibroblasts include, but are notlimited to, synovial fibroblasts. Immune cells may include CD8+ and CD4+T cells, NK cells, B cells, macrophages, and megakaryocytes. IL-22activity can be determined in vitro, for example, using the IL-22receptor inhibition assay as described in Examples 2 and 6, the GROasecretion assay in Example 9, or the BAF3 proliferation assay of Example7. IL-22 activity can also be determined in vivo, for example, byscoring progression of an immune response or disorder as described inExample 13.

As used herein, “in vitro generated antibody” refers to an antibodywhere all or part of the variable region (e.g., at least one CDR) isgenerated in a non-immune cell selection (e.g., an in vitro phagedisplay, protein chip or any other method in which candidate sequencescan be tested for their ability to bind to an antigen). This termexcludes sequences generated by genomic rearrangement in an immune cell.

The term “isolated” refers to a molecule that is substantially free ofits natural environment. For instance, an isolated protein issubstantially free of cellular material or other proteins from the cellor tissue source from which it was derived. The term also refers topreparations where the isolated protein is sufficiently pure forpharmaceutical compositions; or at least 70-80% (w/w) pure; or at least80-90% (w/w) pure; or at least 90-95% pure; or at least 95%, 96%, 97%,98%, 99%, or 100% (w/w) pure.

The phrase “percent identical” or “percent identity” refers to thesimilarity between at least two different sequences. This percentidentity can be determined by standard alignment algorithms, forexample, the Basic Local Alignment Tool (BLAST) described by Altshul etal. ((1990) J. Mol. Biol., 215: 403-410); the algorithm of Needleman etal. ((1970) J. Mol. Biol., 48: 444-453); or the algorithm of Meyers etal. ((1988) Comput. Appl. Biosci., 4: 11-17). A set of parameters may bethe Blosum 62 scoring matrix with a gap penalty of 12, a gap extendpenalty of 4, and a frameshift gap penalty of 5. The percent identitybetween two amino acid or nucleotide sequences can also be determinedusing the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17)which has been incorporated into the ALIGN program (version 2.0), usinga PAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity is usually calculated by comparingsequences of similar length.

The term “repertoire” refers to at least one nucleotide sequence derivedwholly or partially from at least one sequence encoding at least oneimmunoglobulin. The sequence(s) may be generated by rearrangement invivo of the V, D, and J segments of heavy chains, and the V and Jsegments of light chains. Alternatively, the sequence(s) can begenerated from a cell in response to which rearrangement occurs, e.g.,in vitro stimulation. Alternatively, part or all of the sequence(s) maybe obtained by DNA splicing, nucleotide synthesis, mutagenesis, andother methods, see, e.g., U.S. Pat. No. 5,565,332. A repertoire mayinclude only one sequence or may include a plurality of sequences,including ones in a genetically diverse collection.

The terms “specific binding” or “specifically binds” refers to twomolecules forming a complex that is relatively stable under physiologicconditions. Specific binding is characterized by a high affinity and alow to moderate capacity as distinguished from nonspecific binding whichusually has a low affinity with a moderate to high capacity. Typically,binding is considered specific when the association constant K_(A) ishigher than 10⁶ M⁻¹. If necessary, nonspecific binding can be reducedwithout substantially affecting specific binding by varying the bindingconditions. The appropriate binding conditions, such as concentration ofantibodies, ionic strength of the solution, temperature, time allowedfor binding, concentration of a blocking agent (e.g., serum albumin,milk casein), etc., may be optimized by a skilled artisan using routinetechniques. Illustrative conditions are set forth in Example 3, butother conditions known to the person of ordinary skill in the art fallwithin the scope of this invention.

As used herein, the term “stringent” describes conditions forhybridization and washing. Stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous andnonaqueous methods are described in that reference and either can beused. One example of stringent hybridization conditions is hybridizationin 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed byat least one wash in 0.2×SSC, 0.1% SDS at 50° C. A second example ofstringent hybridization conditions is hybridization in 6×SSC at about45° C., followed by at least one wash in 0.2×SSC, 0.1% SDS at 55° C.Another example of stringent hybridization conditions is hybridizationin 6×SSC at about 45° C., followed by at least one wash in 0.2×SSC, 0.1%SDS at 60° C. A further example of stringent hybridization conditions ishybridization in 6×SSC at about 45° C., followed by at least one wash in0.2×SSC, 0.1% SDS at 65° C. High stringent conditions includehybridization in 0.5M sodium phosphate, 7% SDS at 65° C., followed by atleast one wash at 0.2×SSC, 1% SDS at 65° C.

The phrase “substantially as set out,” “substantially identical” or“substantially homologous” means that the relevant amino acid ornucleotide sequence (e.g., CDR(s), V_(H), or V_(L) domain) will beidentical to or have insubstantial differences (through conserved aminoacid substitutions) in comparison to the sequences which are set out.Insubstantial differences include minor amino acid changes, such as 1 or2 substitutions in a 5 amino acid sequence of a specified region. In thecase of antibodies, the second antibody has the same specificity and hasat least 50% of the affinity of the first antibody.

Sequences substantially identical or homologous (e.g., at least about85% sequence identity) to the sequences disclosed herein are also partof this application. In some embodiment, the sequence identity can beabout 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively,substantial identity or homology exists when the nucleic acid segmentswill hybridize under selective hybridization conditions (e.g., highlystringent hybridization conditions), to the complement of the strand.The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form.

The term “therapeutic agent” is a substance that treats or assists intreating a medical disorder. Therapeutic agents may include, but are notlimited to, substances that modulate immune cells or immune responses ina manner that complements the IL-22 activity of anti-IL-22 antibodies.Non-limiting examples and uses of therapeutic agents are describedherein.

As used herein, a “therapeutically effective amount” of an anti-IL-22antibody refers to an amount of an antibody which is effective, uponsingle or multiple dose administration to a subject (such as a humanpatient) at treating, preventing, curing, delaying, reducing theseverity of, and/or ameliorating at least one symptom of a disorder orrecurring disorder, or prolonging the survival of the subject beyondthat expected in the absence of such treatment.

The term “treatment” refers to a therapeutic or preventative measure.The treatment may be administered to a subject having a medical disorderor who ultimately may acquire the disorder, in order to prevent, cure,delay, reduce the severity of, and/or ameliorate one or more symptoms ofa disorder or recurring disorder, or in order to prolong the survival ofa subject beyond that expected in the absence of such treatment.

II. Anti-IL-22 Antibodies

The disclosure provides novel anti-IL-22 antibodies that comprise novelantigen-binding fragments.

Numerous methods known to those skilled in the art are available forobtaining antibodies or antigen-binding fragments thereof. For example,antibodies can be produced using recombinant DNA methods (U.S. Pat. No.4,816,567). Monoclonal antibodies may also be produced by generation ofhybridomas (see e.g., Kohler and Milstein (1975) Nature, 256: 495-499)in accordance with known methods. Hybridomas formed in this manner arethen screened using standard methods, such as enzyme-linkedimmunosorbent assay (ELISA) and surface plasmon resonance (BIACORE™)analysis, to identify one or more hybridomas that produce an antibodythat specifically binds with a specified antigen. Any form of thespecified antigen may be used as the immunogen, e.g., recombinantantigen, naturally occurring forms, any variants or fragments thereof,as well as antigenic peptide thereof.

One exemplary method of making antibodies includes screening proteinexpression libraries, e.g., phage or ribosome display libraries. Phagedisplay is described, for example, in Ladner et al., U.S. Pat. No.5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al. (1991)Nature, 352: 624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597WO92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO92/01047; WO 92/09690; and WO 90/02809.

In addition to the use of display libraries, the specified antigen canbe used to immunize a non-human animal, e.g., a rodent, e.g., a mouse,hamster, or rat. In one embodiment, the non-human animal includes atleast a part of a human immunoglobulin gene. For example, it is possibleto engineer mouse strains deficient in mouse antibody production withlarge fragments of the human Ig loci. Using the hybridoma technology,antigen-specific monoclonal antibodies derived from the genes with thedesired specificity may be produced and selected. See, e.g., XENOMOUSE™,Green et al. (1994) Nature Genetics 7:13-21, US 2003-0070185, WO96/34096, published Oct. 31, 1996, and PCT Application No.PCT/US96/05928, filed Apr. 29, 1996.

In another embodiment, a monoclonal antibody is obtained from thenon-human animal, and then modified, e.g., humanized, deimmunized,chimeric, may be produced using recombinant DNA techniques known in theart. A variety of approaches for making chimeric antibodies have beendescribed. See e.g., Morrison et al., Proc. Natl. Acad. Sci. U.S.A.81:6851, 1985; Takeda et al., Nature 314:452, 1985, Cabilly et al., U.S.Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi etal., European Patent Publication EP171496; European Patent Publication0173494, United Kingdom Patent GB 2177096B. Humanized antibodies mayalso be produced, for example, using transgenic mice that express humanheavy and light chain genes, but are incapable of expressing theendogenous mouse immunoglobulin heavy and light chain genes. Winterdescribes an exemplary CDR-grafting method that may be used to preparethe humanized antibodies described herein (U.S. Pat. No. 5,225,539). Allof the CDRs of a particular human antibody may be replaced with at leasta portion of a non-human CDR, or only some of the CDRs may be replacedwith non-human CDRs. It is only necessary to replace the number of CDRsrequired for binding of the humanized antibody to a predeterminedantigen.

Humanized antibodies or fragments thereof can be generated by replacingsequences of the F_(v) variable domain that are not directly involved inantigen binding with equivalent sequences from human F_(v) variabledomains. Exemplary methods for generating humanized antibodies orfragments thereof are provided by Morrison (1985) Science 229:1202-1207;by Oi et al. (1986) BioTechniques 4:214; and by U.S. Pat. No. 5,585,089;U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; U.S. Pat. No.5,859,205; and U.S. Pat. No. 6,407,213. Those methods include isolating,manipulating, and expressing the nucleic acid sequences that encode allor part of immunoglobulin Fv variable domains from at least one of aheavy or light chain. Such nucleic acids may be obtained from ahybridoma producing an antibody against a predetermined target, asdescribed above, as well as from other sources. The recombinant DNAencoding the humanized antibody molecule can then be cloned into anappropriate expression vector.

In certain embodiments, a humanized antibody is optimized by theintroduction of conservative substitutions, consensus sequencesubstitutions, germline substitutions and/or backmutations. Such alteredimmunoglobulin molecules can be made by any of several techniques knownin the art, (e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80:7308-7312, 1983; Kozbor et al., Immunology Today, 4: 7279, 1983; Olssonet al., Meth. Enzymol., 92: 3-16, 1982), and may be made according tothe teachings of PCT Publication WO92/06193 or EP 0239400).

An antibody or fragment thereof may also be modified by specificdeletion of human T cell epitopes or “deimmunization” by the methodsdisclosed in WO 98/52976 and WO 00/34317. Briefly, the heavy and lightchain variable domains of an antibody can be analyzed for peptides thatbind to MHC Class II; these peptides represent potential T-cell epitopes(as defined in WO 98/52976 and WO 00/34317). For detection of potentialT-cell epitopes, a computer modeling approach termed “peptide threading”can be applied, and in addition a database of human MHC class II bindingpeptides can be searched for motifs present in the V_(H) and V_(L)sequences, as described in WO 98/52976 and WO 00/34317. These motifsbind to any of the 18 major MHC class II DR allotypes, and thusconstitute potential T cell epitopes. Potential T-cell epitopes detectedcan be eliminated by substituting small numbers of amino acid residuesin the variable domains, or preferably, by single amino acidsubstitutions. Typically, conservative substitutions are made. Often,but not exclusively, an amino acid common to a position in humangermline antibody sequences may be used. Human germline sequences, e.g.,are disclosed in Tomlinson, et al. (1992) J. Mol. Biol. 227:776-798;Cook, G. P. et al. (1995) Immunol. Today Vol. 16 (5): 237-242; Chothia,D. et al. (1992) J. Mol. Biol. 227:799-817; and Tomlinson et al. (1995)EMBO J. 14:4628-4638. The V BASE directory provides a comprehensivedirectory of human immunoglobulin variable region sequences (compiled byTomlinson, I. A. et al., MRC Centre for Protein Engineering, Cambridge,UK). These sequences can be used as a source of human sequence, e.g.,for framework regions and CDRs. Consensus human framework regions canalso be used, e.g., as described in U.S. Pat. No. 6,300,064.

In certain embodiments, an antibody can contain an alteredimmunoglobulin constant or Fc region. For example, an antibody producedin accordance with the teachings herein may bind more strongly or withmore specificity to effector molecules such as complement and/or Fcreceptors, which can control several immune functions of the antibodysuch as effector cell activity, lysis, complement-mediated activity,antibody clearance, and antibody half-life. Typical Fc receptors thatbind to an Fc region of an antibody (e.g., an IgG antibody) include, butare not limited to, receptors of the FcγRI, FcγRII, and FcγRIII and FcRnsubclasses, including allelic variants and alternatively spliced formsof these receptors. Fc receptors are reviewed in Ravetch and Kinet,Annu. Rev. Immunol 9:457-92, 1991; Capel et al., Immunomethods 4:25-34,1994; and de Haas et al., J. Lab. Clin. Med. 126:330-41, 1995).

For additional antibody production techniques, see Antibodies: ALaboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory,1988. The present invention is not necessarily limited to any particularsource, method of production, or other special characteristics of anantibody.

Antibodies, also known as immunoglobulins, are typically tetramericglycosylated proteins composed of two light (L) chains of approximately25 kDa each and two heavy (H) chains of approximately 50 kDa each. Twotypes of light chain, termed lambda and kappa, may be found inantibodies. Depending on the amino acid sequence of the constant domainof heavy chains, immunoglobulins can be assigned to five major classes:A, D, E, G, and M, and several of these may be further divided intosubclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂.Each light chain includes an N-terminal variable (V) domain (VL) and aconstant (C) domain (CL). Each heavy chain includes an N-terminal Vdomain (VH), three or four C domains (CHs), and a hinge region. The CHdomain most proximal to VH is designated as CH1. The VH and VL domainsconsist of four regions of relatively conserved sequences calledframework regions (FR1, FR2, FR3, and FR4), which form a scaffold forthree regions of hypervariable sequences (complementarity determiningregions, CDRs). The CDRs contain most of the residues responsible forspecific interactions of the antibody with the antigen. CDRs arereferred to as CDR1, CDR2, and CDR3. Accordingly, CDR constituents onthe heavy chain are referred to as H1, H2, and H3, while CDRconstituents on the light chain are referred to as L1, L2, and L3.

CDR3 is typically the greatest source of molecular diversity within theantibody-binding site. H3, for example, can be as short as two aminoacid residues or greater than 26 amino acids. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known in the art. For a review of the antibody structure, seeAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds.Harlow et al., 1988. One of skill in the art will recognize that eachsubunit structure, e.g., a CH, VH, CL, VL, CDR, FR structure, comprisesactive fragments, e.g., the portion of the VH, VL, or CDR subunit thebinds to the antigen, i.e., the antigen-binding fragment, or, e.g., theportion of the CH subunit that binds to and/or activates, e.g., an Fcreceptor and/or complement. The CDRs typically refer to the Kabat CDRs,as described in Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services (1991), eds. Kabat et al.Another standard for characterizing the antigen binding site is to referto the hypervariable loops as described by Chothia. See, e.g., Chothia,D. et al. (1992) J. Mol. Biol. 227:799-817; and Tomlinson et al. (1995)EMBO J. 14:4628-4638. Still another standard is the AbM definition usedby Oxford Molecular's AbM antibody modelling software. See, generally,e.g., Protein Sequence and Structure Analysis of Antibody VariableDomains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. andKontermann, R., Springer-Verlag, Heidelberg). Embodiments described withrespect to Kabat CDRs can alternatively be implemented using similardescribed relationships with respect to Chothia hypervariable loops orto the AbM-defined loops.

The Fab fragment (Fragment antigen-binding) consists of V_(H)-C_(H)1 andV_(L)-C_(L) domains covalently linked by a disulfide bond between theconstant regions. The F_(v) fragment is smaller and consists of V_(H)and V_(L) domains non-covalently linked. To overcome the tendency ofnon-covalently linked domains to dissociate, a single chain F_(v)fragment (scFv) can be constructed. The scF_(v) contains a flexiblepolypeptide that links (1) the C-terminus of V_(H) to the N-terminus ofV_(L), or (2) the C-terminus of V_(L) to the N-terminus of V_(H). A15-mer (Gly₄Ser)₃ peptide may be used as a linker, but other linkers areknown in the art.

The sequence of antibody genes after assembly and somatic mutation ishighly varied, and these varied genes are estimated to encode 10¹⁰different antibody molecules (Immunoglobulin Genes, 2nd ed., eds. Jonioet al., Academic Press, San Diego, Calif. 1995).

A “bispecific” or “bifunctional antibody” is an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. Bispecific antibodies can be produced by a variety ofmethods including fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321(1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992). In oneembodiment, the bispecific antibody comprises a first binding domainpolypeptide, such as a Fab′ fragment, linked via an immunoglobulinconstant region to a second binding domain polypeptide.

Small Modular ImmunoPharmaceuticals (SMIP™) provide an example of avariant molecule comprising a binding domain polypeptide. SMIPs andtheir uses and applications are disclosed in, e.g., U.S. PublishedPatent Application. Nos. 2003/0118592, 2003/0133939, 2004/0058445,2005/0136049, 2005/0175614, 2005/0180970, 2005/0186216, 2005/0202012,2005/0202023, 2005/0202028, 2005/0202534, and 2005/0238646, and relatedpatent family members thereof, all of which are hereby incorporated byreference herein in their entireties.

A SMIP™ typically refers to a binding domain-immunoglobulin fusionprotein that includes a binding domain polypeptide that is fused orotherwise connected to an immunoglobulin hinge or hinge-acting regionpolypeptide, which in turn is fused or otherwise connected to a regioncomprising one or more native or engineered constant regions from animmunoglobulin heavy chain, other than CH1, for example, the CH2 and CH3regions of IgG and IgA, or the CH3 and CH4 regions of IgE (see e.g.,U.S. 2005/0136049 by Ledbetter, J. et al., which is incorporated byreference, for a more complete description). The bindingdomain-immunoglobulin fusion protein can further include a region thatincludes a native or engineered immunoglobulin heavy chain CH2 constantregion polypeptide (or CH3 in the case of a construct derived in wholeor in part from IgE) that is fused or otherwise connected to the hingeregion polypeptide and a native or engineered immunoglobulin heavy chainCH3 constant region polypeptide (or CH4 in the case of a constructderived in whole or in part from IgE) that is fused or otherwiseconnected to the CH2 constant region polypeptide (or CH3 in the case ofa construct derived in whole or in part from IgE). Typically, suchbinding domain-immunoglobulin fusion proteins are capable of at leastone immunological activity selected from the group consisting ofantibody dependent cell-mediated cytotoxicity, complement fixation,and/or binding to a target, for example, a target antigen, such as humanIL-22.

Therapeutic proteins, i.e., a protein or peptide that has a biologicaleffect on a region in the body on which it acts or on a region of thebody on which it remotely acts via intermediates, are also useful forpracticing the invention. A therapeutic protein can include peptidemimetics. Mimetics are peptide-containing molecules that mimic elementsof protein secondary structure. See, for example, Johnson et al.,“Peptide Turn Mimetics” in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al.,Eds., Chapman and Hall, New York (1993), incorporated herein byreference. The underlying rationale behind the use of peptide mimeticsis that the peptide backbone of proteins exists chiefly to orient aminoacid side chains in such a way as to facilitate molecular interactions,such as those of antibody and antigen. A peptide mimetic is expected topermit molecular interactions similar to the natural molecule. Theseprinciples may be used to engineer second generation molecules havingmany of the natural properties of the targeting peptides disclosedherein, but with altered and potentially improved characteristics.

Other embodiments of therapeutic proteins include fusion proteins. Thesemolecules generally have all or a substantial portion of a targetingpeptide, for example, IL-22 or an anti-IL-22 antibody, linked at the N-or C-terminus, to all or a portion of a second polypeptide or protein.For example, fusions may employ leader sequences from other species topermit the recombinant expression of a protein in a heterologous host.Another useful fusion includes the addition of an immunologically activedomain, such as an antibody epitope, to facilitate purification of thefusion protein. Inclusion of a cleavage site at or near the fusionjunction will facilitate removal of the extraneous polypeptide afterpurification. Other useful fusions include linking of functionaldomains, such as active sites from enzymes, glycosylation domains,cellular targeting signals or transmembrane regions. Examples ofproteins or peptides that may be incorporated into a fusion proteininclude cytostatic proteins, cytocidal proteins, pro-apoptosis agents,anti-angiogenic agents, hormones, cytokines, growth factors, peptidedrugs, antibodies, Fab fragments of antibodies, antigens, receptorproteins, enzymes, lectins, MHC proteins, cell adhesion proteins andbinding proteins. Methods of generating fusion proteins are well knownto those of skill in the art. Such proteins can be produced, forexample, by chemical attachment using bifunctional cross-linkingreagents, by de novo synthesis of the complete fusion protein, or byattachment of a DNA sequence encoding the targeting peptide to a DNAsequence encoding the second peptide or protein, followed by expressionof the intact fusion protein.

In one embodiment, the targeting peptide, for example, IL-22 or ananti-IL-22 antibody, is fused with an immunoglobulin heavy chainconstant region, such as an Fc fragment, which contains two constantregion domains and a hinge region but lacks the variable region (See,U.S. Pat. Nos. 6,018,026 and 5,750,375, incorporated herein byreference). The Fc region may be a naturally occurring Fc region, or maybe altered to improve certain qualities, such as therapeutic qualities,circulation time, reduced aggregation, etc. Peptides and proteins fusedto an Fc region typically exhibit a greater half-life in vivo than theunfused counterpart. Also, a fusion to an Fc region permitsdimerization/multimerization of the fusion polypeptide.

VHH molecules (or nanobodies), as known to the skilled artisan, areheavy chain variable domains derived from immunoglobulins naturallydevoid of light chains, such as those derived from Camelidae asdescribed in WO 9404678, incorporated herein by reference. Such a VHHmolecule can be derived from antibodies raised in Camelidae species, forexample in camel, llama, dromedary, alpaca and guanaco and is sometimescalled a camelid or camelized variable domain. See e.g., Muyldermans.,J. Biotechnology (2001) 74(4):277-302, incorporated herein by reference.Other species besides Camelidae may produce heavy chain antibodiesnaturally devoid of light chain. VHH molecules are about 10 timessmaller than IgG molecules. They are single polypeptides and verystable, resisting extreme pH and temperature conditions. Moreover, theyare resistant to the action of proteases which is not the case forconventional antibodies. Furthermore, in vitro expression of VHHsproduces high yield, properly folded functional VHHs. In addition,antibodies generated in Camelids will recognize epitopes other thanthose recognized by antibodies generated in vitro through the use ofantibody libraries or via immunization of mammals other than Camelids(see WO 9749805, which is incorporated herein by reference).

One aspect of the present invention comprises antibodies and antigenbinding fragments that bind IL-22. The disclosure provides novel CDRsderived from human immunoglobulin gene libraries. The structure forcarrying a CDR is generally an antibody heavy or light chain or portionthereof, where the CDR is located to a naturally occurring CDR region.The structures and locations of variable domains may be determined asdescribed in Kabat et al., Sequences of Proteins of ImmunologicalInterest, No. 91-3242, National Institutes of Health Publications,Bethesda, Md. (1991).

DNA and amino acid (AA) sequences of illustrative embodiments of theanti-IL-22 antibodies of this invention, including their scF_(v)fragments, V_(H) and V_(L) domains, and CDRs, are set forth in FIGS.7-10 and enumerated in Tables 1 and 7. Twenty specific embodiments ofthe non-germlined antibodies are identified as GIL01, GIL16, GIL45,GIL60, GIL68, GIL92, 097D09, 062A09, 062G05, 087B03, 367D04, 368D04,166B06, 166G05, 375G06, 376B10, 354A08, 355B06, 355E04, and 356A11. TheCDR positions in the V_(H) and V_(L) domains of the non-germlinedantibodies are listed in Table 2. Fifteen specific embodiments of thegermlined antibodies are identified as GIL01, GIL16, GIL45, GIL60,GIL68, GIL92, 062A09, 087B03, 166B06, 166G05, 354A08, 355B06, 355E04,356A11, and 368D04.

TABLE 1A Amino acid and Nucleotide Sequences of V_(H) and V_(L) Domains,Fv, and CDRs of Non-germlined Antibodies GIL01 GIL16 GIL45 GIL60 GIL68GIL92 097D09 062A09 062G05 087B03 Region Type SEQ ID SEQ ID SEQ ID SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID V_(H) AA NO: 5 NO: 23 NO:41 NO: 59 NO: 77 NO: 95 NO: 113 NO: 131 NO: 149 NO: 167 V_(L) AA NO: 6NO: 24 NO: 42 NO: 60 NO: 78 NO: 96 NO: 114 NO: 132 NO: 150 NO: 168 scFvAA NO: 7 NO: 25 NO: 43 NO: 61 NO: 79 NO: 97 NO: 115 NO: 133 NO: 151 NO:169 H1 AA NO: 8 NO: 26 NO: 44 NO: 62 NO: 80 NO: 98 NO: 116 NO: 134 NO:152 NO: 170 H2 AA NO: 9 NO: 27 NO: 45 NO: 63 NO: 81 NO: 99 NO: 117 NO:135 NO: 153 NO: 171 H3 AA NO: 10 NO: 28 NO: 46 NO: 64 NO: 82 NO: 100 NO:118 NO: 136 NO: 154 NO: 172 L1 AA NO: 11 NO: 29 NO: 47 NO: 65 NO: 83 NO:101 NO: 119 NO: 137 NO: 155 NO: 173 L2 AA NO: 12 NO: 30 NO: 48 NO: 66NO: 84 NO: 102 NO: 120 NO: 138 NO: 156 NO: 174 L3 AA NO: 13 NO: 31 NO:49 NO: 67 NO: 85 NO: 103 NO: 121 NO: 139 NO: 157 NO: 175 V_(H) DNA NO:14 NO: 32 NO: 50 NO: 68 NO: 86 NO: 104 NO: 122 NO: 140 NO: 158 NO: 176V_(L) DNA NO: 15 NO: 33 NO: 51 NO: 69 NO: 87 NO: 105 NO: 123 NO: 141 NO:159 NO: 177 scF_(v) DNA NO: 16 NO: 34 NO: 52 NO: 70 NO: 88 NO: 106 NO:124 NO: 142 NO: 160 NO: 178 H1 DNA NO: 17 NO: 35 NO: 53 NO: 71 NO: 89NO: 107 NO: 125 NO: 143 NO: 161 NO: 179 H2 DNA NO: 18 NO: 36 NO: 54 NO:72 NO: 90 NO: 108 NO: 126 NO: 144 NO: 162 NO: 180 H3 DNA NO: 19 NO: 37NO: 55 NO: 73 NO: 91 NO: 109 NO: 127 NO: 145 NO: 163 NO: 181 L1 DNA NO:20 NO: 38 NO: 56 NO: 74 NO: 92 NO: 110 NO: 128 NO: 146 NO: 164 NO: 182L2 DNA NO: 21 NO: 39 NO: 57 NO: 75 NO: 93 NO: 111 NO: 129 NO: 147 NO:165 NO: 183 L3 DNA NO: 22 NO: 40 NO: 58 NO: 76 NO: 94 NO: 112 NO: 130NO: 148 NO: 166 NO: 184

TABLE 1B Amino acid and Nucleotide Sequences of V_(H) and V_(L) Domains,Fv, and CDRs of Non-germlined Antibodies 367D04 368D04 166B06 166G05375G06 376B10 354A08 355B06 355E04 356A11 Region Type SEQ ID SEQ ID SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID V_(H) AA NO: 185 NO:203 NO: 221 NO: 239 NO: 257 NO: 275 NO: 293 NO: 311 NO: 329 NO: 347V_(L) AA NO: 186 NO: 204 NO: 222 NO: 240 NO: 258 NO: 276 NO: 294 NO: 312NO: 330 NO: 348 scF_(v) AA NO: 187 NO: 205 NO: 223 NO: 241 NO: 259 NO:277 NO: 295 NO: 313 NO: 331 NO: 349 H1 AA NO: 188 NO: 206 NO: 224 NO:242 NO: 260 NO: 278 NO: 296 NO: 314 NO: 332 NO: 350 H2 AA NO: 189 NO:207 NO: 225 NO: 243 NO: 261 NO: 279 NO: 297 NO: 315 NO: 333 NO: 351 H3AA NO: 190 NO: 208 NO: 226 NO: 244 NO: 262 NO: 280 NO: 298 NO: 316 NO:334 NO: 352 L1 AA NO: 191 NO: 209 NO: 227 NO: 245 NO: 263 NO: 281 NO:299 NO: 317 NO: 335 NO: 353 L2 AA NO: 192 NO: 210 NO: 228 NO: 246 NO:264 NO: 282 NO: 300 NO: 318 NO: 336 NO: 354 L3 AA NO: 193 NO: 211 NO:229 NO: 247 NO: 265 NO: 283 NO: 301 NO: 319 NO: 337 NO: 355 V_(H) DNANO: 194 NO: 212 NO: 230 NO: 248 NO: 266 NO: 284 NO: 302 NO: 320 NO: 338NO: 356 V_(L) DNA NO: 195 NO: 213 NO: 231 NO: 249 NO: 267 NO: 285 NO:303 NO: 321 NO: 339 NO: 357 scF_(v) DNA NO: 196 NO: 214 NO: 232 NO: 250NO: 268 NO: 286 NO: 304 NO: 322 NO: 340 NO: 358 H1 DNA NO: 197 NO: 215NO: 233 NO: 251 NO: 269 NO: 287 NO: 305 NO: 323 NO: 341 NO: 359 H2 DNANO: 198 NO: 216 NO: 234 NO: 252 NO: 270 NO: 288 NO: 306 NO: 324 NO: 342NO: 360 H3 DNA NO: 199 NO: 217 NO: 235 NO: 253 NO: 271 NO: 289 NO: 307NO: 325 NO: 343 NO: 361 L1 DNA NO: 200 NO: 218 NO: 236 NO: 254 NO: 272NO: 290 NO: 308 NO: 326 NO: 344 NO: 362 L2 DNA NO: 201 NO: 219 NO: 237NO: 255 NO: 273 NO: 291 NO: 309 NO: 327 NO: 345 NO: 363 L3 DNA NO: 202NO: 220 NO: 238 NO: 256 NO: 274 NO: 292 NO: 310 NO: 328 NO: 346 NO: 364

TABLE 2 Positions of CDRs within Non-germlined Antibody Amino AcidSequences of V_(H) and V_(L) Domains CDR GIL01 GIL16 GIL45 GIL60 GIL68GIL92 097D09 062A09 062G05 087B03 H1 31-35 31-35 31-35 31-35 31-35 31-3531-35 31-35 31-35 31-35 H2 50-66 50-66 50-66 50-66 50-66 50-66 50-6650-66 50-66 50-66 H3  99-108  99-108  99-108  99-110  99-108  99-110 99-108  99-108  99-108  99-110 L1 24-34 24-34 23-36 23-36 23-33 23-3624-34 24-34 24-34 23-36 L2 50-56 50-56 52-58 52-58 49-55 52-58 50-5650-56 50-56 52-58 L3 89-97 89-97  91-100  91-100 88-98  91-101 89-9789-97 89-97  91-100 CDR 367D04 368D04 166B06 166G05 375G06 376B10 354A08355B06 355E04 356A11 H1 31-35 31-35 31-35 31-35 31-35 31-35 30-34 31-3531-35 31-35 H2 50-66 50-66 50-66 50-66 50-66 50-66 49-65 50-66 50-6650-66 H3  99-110  99-110  99-108  99-108  99-108  99-108  98-109  99-110 99-110  99-110 L1 23-36 23-36 23-33 23-33 23-33 23-33 23-36 23-36 23-3622-35 L2 52-58 52-58 49-55 49-55 49-55 49-55 52-58 52-58 52-58 51-58 L3 91-100  91-100 88-98 88-98 88-98 88-98  91-101  91-101  91-101  90-100

Anti-IL-22 antibodies of this invention may optionally comprise antibodyconstant regions or parts thereof. For example, a V_(L) domain may beattached at its C-terminal end to a light chain constant domain like Cκor Cλ. Similarly, a V_(H) domain or portion thereof may be attached toall or part of a heavy chain like IgA, IgD, IgE, IgG, and IgM, and anyisotype subclass. Constant regions are known in the art (see, forexample, Kabat et al., Sequences of Proteins of Immunological Interest,No. 91-3242, National Institutes of Health Publications, Bethesda, Md.(1991)). Therefore, antibodies within the scope of this inventioninclude V_(H) and V_(L) domains, or a portion thereof, combined withconstant regions known in the art.

Certain embodiments comprise a V_(H) domain, a V_(L) domain, or acombination thereof, of the F_(v) fragment from GIL01, GIL16, GIL45,GIL60, GIL68, GIL92, 097D09, 062A09, 062G05, 087B03, 367D04, 368D04,166B06, 166G05, 375G06, 376B10, 354A08, 355B06, 355E04, or 356A11.Another embodiment comprises a V_(H) domain, a V_(L) domain, or acombination thereof, of the F_(v) fragment from an antibody chosen from356A11, 354A08, 087B03, and 368D04. Further embodiments comprise one,two, three, four, five or six complementarity determining regions (CDRs)from the V_(H) and V_(L) domains. Antibodies whose CDR sequences areincluded within SEQ ID NO:5-13, 23-31, 41-49, 59-67, 77-85, 95-103,113-121, 131-139, 149-157, 167-175, 185-193, 203-211, 221-229, 239-247,257-265, 275-283, 293-301, 311-319, 329-337, 347-355, 365-373, 383-391,401-409, 419-427, 437-445, 455-463, 473-481, 491-499, 509-517, 527-535,545-553, 563-571, 581-589, 599-607, or 617-625 are encompassed withinthe scope of this invention. For example, in one embodiment, an antibodycomprises a H3 fragment of the V_(H) domain of germlined ornon-germlined GIL01, GIL16, GIL45, GIL60, GIL68, GIL92, 097D09, 062A09,062G05, 087B03, 367D04, 368D04, 166B06, 166G05, 375G06, 376B10, 354A08,355B06, 355E04, or 356A11 or from an antibody chosen from 356A11,354A08, 087B03, and 368D04.

In certain embodiments, the V_(H) and/or V_(L) domains may be germlined,i.e., the framework regions (FR) of these domains are mutated usingconventional molecular biology techniques to match those produced by thegermline cells. In other embodiments, the FR sequences remain divergedfrom the consensus germline sequences. In one embodiment of thisinvention, germlined antibodies are shown in Table 7.

In one embodiment, the invention provides amino acid and nucleic acidsequences for the germlined GIL01, GIL16, GIL45, GIL60, GIL68, GIL92,097D09, 062A09, 062G05, 087B03, 367D04, 368D04, 166B06, 166G05, 375G06,376B10, 354A08, 355B06, 355E04, or 356A11. Amino acid and nucleotidesequences for the V_(H) domain of the germlined GIL01, GIL16, GIL45,GIL60, GIL68, GIL92, 062A09, 087B03, 166B06, 166G05, 354A08, 355B06,355E04, 356A11, and 368D04 are depicted in Table 7 and FIG. 8. Aminoacid and nucleotide sequences for the V_(L) domain of the germlinedGIL01, GILL 6, GIL45, GIL60, GIL68, GIL92, 062A09, 087B03, 166B06,166G05, 354A08, 355B06, 355E04, 356A11, and 368D04 are also depicted inTable 7 and FIG. 8.

In one embodiment, mutagenesis is used to make an antibody more similarto one or more germline sequences. This may be desirable when mutationsare introduced into the framework region of an antibody through somaticmutagenesis or through error prone PCR. Germline sequences for the V_(H)and V_(L) domains can be identified by performing amino acid and nucleicacid sequence alignments against the VBASE database (MRC Center forProtein Engineering, UK). VBASE is a comprehensive directory of allhuman germline variable region sequences compiled from over a thousandpublished sequences, including those in the current releases of theGenbank and EMBL data libraries. In some embodiments, the FR regions ofthe scFvs are mutated in conformity with the closest matches in theVBASE database and the CDR portions are kept intact.

In certain embodiments, antibodies of this invention specifically reactwith an epitope that is the same as the epitope recognized by GIL01,GIL16, GIL45, GIL60, GIL68, GIL92, 097D09, 062A09, 062G05, 087B03,367D04, 368D04, 166B06, 166G05, 375G06, 376B10, 354A08, 355B06, 355E04,or 356A11, such that they competitively inhibit the binding of GIL01,GIL16, GIL45, GIL60, GIL68, GIL92, 097D09, 062A09, 062G05, 087B03,367D04, 368D04, 166B06, 166G05, 375G06, 376B10, 354A08, 355B06, 355E04,or 356A11 to human IL-22. Such antibodies can be determined incompetitive binding assays. In one embodiment, the antibody, or antigenbinding fragment thereof, binds to an IL-22 epitope that is recognizedby 368D04, such that the antibody competitively inhibits the binding of368D04 to human IL-22. In another embodiment, the antibody, or antigenbinding fragment thereof, binds to an IL-22 epitope that is recognizedby 356A11, such that the antibody competitively inhibits the binding of356A11 to human IL-22. In another embodiment, the antibody, or antigenbinding fragment thereof, binds to an IL-22 epitope that is recognizedby 354A08, such that the antibody competitively inhibits the binding of354A08 to human IL-22. In another embodiment, the antibody, or antigenbinding fragment thereof, binds to an IL-22 epitope that is recognizedby 087B03, such that the antibody competitively inhibits the binding of087B03 to human IL-22. In one embodiment, the association constant(K_(A)) of these antibodies for human IL-22 is at least 10⁶ M⁻. Inanother embodiment, the association constant of these antibodies forhuman IL-22 is at least 10⁹ M⁻¹. In other embodiments, the associationconstant of these antibodies for human IL-22 is at least 10¹⁰ M⁻¹, atleast 10¹¹ M⁻¹, or at least 1012 M⁻¹. The binding affinity may bedetermined using techniques known in the art, such as ELISA, biosensortechnology, such as biospecific interaction analysis, or othertechniques including those described in this application.

It is contemplated that antibodies of this invention may bind otherproteins, such as, for example, recombinant proteins comprising all or aportion of IL-22.

One of ordinary skill in the art will recognize that the disclosedantibodies may be used to detect, measure, and/or inhibit proteins thatdiffer somewhat from IL-22. For example, these proteins may be homologsof IL-22. Anti-IL-22 antibodies are expected to bind proteins thatcomprise a sequence which is at least about 60%, 70%, 80%, 90%, 95%, ormore identical to any sequence of at least 100, 80, 60, 40, or 20contiguous amino acids in the sequence set forth SEQ ID NO:1.

In addition to sequence homology analyses, epitope mapping (see, e.g.,Epitope Mapping Protocols, ed. Morris, Humana Press, 1996), andsecondary and tertiary structure analyses can be carried out to identifyspecific 3D structures assumed by the presently disclosed antibodies andtheir complexes with antigens. Such methods include, but are not limitedto, X-ray crystallography (Engstom (1974) Biochem. Exp. Biol., 11:7-13)and computer modeling of virtual representations of the presentantibodies (Fletterick et al. (1986) Computer Graphics and MolecularModeling, in Current Communications in Molecular Biology, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.).

The disclosure provides a method for obtaining anti-IL-22 antibodiesthat comprises creating antibodies with altered Table 1 V_(H) and/orV_(L) sequence(s). Such antibodies may be derived by a skilled artisanusing techniques known in the art. For example, amino acidsubstitutions, deletions, or additions can be introduced in FR and/orCDR regions. FR changes are usually designed to improve the stabilityand immunogenicity of the antibody, while CDR changes are typicallydesigned to increase antibody affinity for its antigen. The changes thatincrease affinity may be tested by altering CDR sequence and measuringantibody affinity for its target (see Antibody Engineering, 2nd ed.,Oxford University Press, ed. Borrebaeck, 1995).

Antibodies whose CDR sequences differ insubstantially from thoseincluded in or included within the sequences in SEQ ID NO: 5-13, 23-31,41-49, 59-67, 77-85, 95-103, 113-121, 131-139, 149-157, 167-175,185-193, 203-211, 221-229, 239-247, 257-265, 275-283, 293-301, 311-319,329-337, 347-355, 365-373, 383-391, 401-409, 419-427, 437-445, 455-463,473-481, 491-499, 509-517, 527-535, 545-553, 563-571, 581-589, 599-607,or 617-625, are encompassed within the scope of this invention.Typically, this involves substitution of an amino acid with an aminoacid having similar charge, hydrophobic, or stereochemicalcharacteristics. More drastic substitutions in FR regions, in contrastto CDR regions, may also be made as long as they do not adversely affect(e.g., reduce affinity by more than 50% as compared to unsubstitutedantibody) the binding properties of the antibody. Substitutions may alsobe made to germline the antibody or stabilize the antigen binding site.

Conservative modifications will produce molecules having functional andchemical characteristics similar to those of the molecule from whichsuch modifications are made. In contrast, substantial modifications inthe functional and/or chemical characteristics of the molecules may beaccomplished by selecting substitutions in the amino acid sequence thatdiffer significantly in their effect on maintaining (1) the structure ofthe molecular backbone in the area of the substitution, for example, asa sheet or helical conformation, (2) the charge or hydrophobicity of themolecule at the target site, or (3) the size of the molecule.

For example, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a normative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position. (See, for example, MacLennan etal., 1998, Acta Physiol. Scand. Suppl. 643:55-67; Sasaki et al., 1998,Adv. Biophys. 35:1-24).

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of the moleculesequence, or to increase or decrease the affinity of the moleculesdescribed herein. Exemplary amino acid substitutions include, but arenot limited to, those set forth in Table 3.

TABLE 3 Amino Acid Substitutions More Original Exemplary ConservativeResidues Substitutions Substitutions Ala (A) Val, Leu, Lle Val Arg (R)Lys, Gln, Asn Lys Asn (N) Gln Gln Asp (D) Glu Glu Cys (C) Ser, Ala SerGln (Q) Asn Asn Gly (G) Pro, Ala Ala His (H) Asn, Gln, Lys, Arg Arg Ile(I) Leu, Val, Met, Ala, Phe, Norleucine Leu Leu (L) Norleucine, Ile,Val, Met, Ala, Phe Ile Lys (K) Arg, 1, 4 Diamino-butyric Acid, Gln, ArgAsn Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro(P) Ala Gly Ser (S) Thr, Ala, Cys Thr Thr (T) Ser Ser Trp (W) Tyr, PheTyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Met, Leu, Phe, Ala,Norleucine Leu

In certain embodiments, conservative amino acid substitutions alsoencompass non-naturally occurring amino acid residues which aretypically incorporated by chemical peptide synthesis rather than bysynthesis in biological systems.

In one embodiment, the method for making a variant V_(H) domaincomprises adding, deleting, or substituting at least one amino acid inthe disclosed V_(H) domains, or combining the disclosed V_(H) domainswith at least one V_(L) domain, and testing the variant V_(H) domain forIL-22 binding or modulation of IL-22 activity.

An analogous method for making a variant V_(L) domain comprises adding,deleting, or substituting at least one amino acid in the disclosed V_(L)domains, or combining the disclosed V_(L) domains with at least oneV_(H) domain, and testing the variant V_(L) domain for IL-22 binding ormodulation of IL-22 activity.

A further aspect of the disclosure provides a method for preparingantibodies or antigen-binding fragments that specifically bind IL-22.The method comprises:

(a) providing a starting repertoire of nucleic acids encoding a V_(H)domain which lacks at least one CDR or contains at least one CDR to bereplaced;

(b) inserting into or replacing the CDR region of the startingrepertoire with at least one donor nucleic acid encoding an amino acidsequence as substantially set out herein for a V_(H) CDR, yielding aproduct repertoire;

(c) expressing the nucleic acids of the product repertoire;

(d) selecting a specific antigen-binding fragment that binds to IL-22;and

(e) recovering the specific antigen-binding fragment or nucleic acidencoding it.

In an analogous method at least one V_(L) CDR of the invention iscombined with a repertoire of nucleic acids encoding a V_(L) domainwhich lacks at least one CDR or contains at least one CDR to bereplaced. The at least one V_(H) or V_(L) CDR may be a CDR1, a CDR2, aCDR3, or a combination thereof, including combinations of V_(H) andV_(L) CDRs, such as those set forth in Tables 1 or 7, including thoseset out in SEQ ID NO:8, 9, 10, 11, 12, 13, 26, 27, 28, 29, 30, 31, 44,45, 46, 47, 48, 49, 62, 63, 64, 65, 66, 67, 80, 81, 82, 83, 84, 85, 98,99, 100, 101, 102, 103, 116, 117, 118, 119, 120, 121, 134, 135, 136,137, 138, 139, 152, 153, 154, 155, 156, 157, 170, 171, 172, 173, 174,175, 188, 189, 190, 191, 192, 193, 206, 207, 208, 209, 210, 211, 224,225, 226, 227, 228, 229, 242, 243, 244, 245, 246, 247, 260, 261, 262,263, 264, 265, 278, 279, 280, 281, 282, 283, 296, 297, 298, 299, 300,301, 314, 315, 316, 317, 318, 319, 332, 333, 334, 335, 336, 337, 350,351, 352, 353, 354, 355, 368, 369, 370, 371, 372, 373, 386, 387, 388,389, 390, 391, 404, 405, 406, 407, 408, 409, 422, 423, 424, 425, 426,427, 440, 441, 442, 443, 444, 445, 458, 459, 460, 461, 462, 463, 476,477, 478, 479, 480, 481, 494, 495, 496, 497, 498, 499, 512, 513, 514,515, 516, 517, 530, 531, 532, 533, 534, 535, 548, 549, 550, 551, 552,553, 566, 567, 568, 569, 570, 571, 584, 585, 586, 587, 588, 589, 602,603, 604, 605, 606, 607, 620, 621, 622, 623, 624, or 625.

In one embodiment, the variable domain includes a CDR3 to be replaced orlacks a CDR3 encoding region and the at least one donor nucleic acidencodes an amino acid substantially as set out in SEQ ID NO:10, 13, 28,31, 46, 49, 64, 67, 82, 85, 100, 103, 118, 121, 136, 139, 154, 157, 172,175, 190, 193, 208, 211, 226, 229, 244, 247, 262, 265, 280, 283, 298,301, 316, 319, 334, 337, 352, 355, 370, 373, 388, 391, 406, 409, 424,427, 442, 445, 460, 463, 478, 481, 496, 499, 514, 517, 532, 535, 550,553, 568, 571, 586, 589, 604, 607, 622, or 625.

In another embodiment, the variable domain includes a CDR1 to bereplaced or lacks a CDR1 encoding region and the at least one donornucleic acid encodes an amino acid sequence substantially as set out inSEQ ID NO:8, 11, 26, 29, 44, 47, 62, 65, 80, 83, 98, 101, 116, 119, 134,137, 152, 155, 170, 173, 188, 191, 206, 209, 224, 227, 242, 245, 260,263, 278, 281, 296, 299, 314, 317, 332, 335, 350, 353, 368, 371, 386,389, 404, 407, 422, 425, 440, 443, 458, 461, 476, 479, 494, 497, 512,515, 530, 533, 548, 551, 566, 569, 584, 587, 602, 605, 620, or 623.

In another embodiment, the variable domain includes a CDR2 to bereplaced or lacks a CDR2 encoding region and the at least one donornucleic acid encodes an amino acid sequence substantially as set out inSEQ ID NO:9, 12, 27, 30, 45, 48, 63, 66, 81, 84, 99, 102, 117, 120, 135,138, 153, 156, 171, 174, 189, 192, 207, 210, 225, 228, 243, 246, 261,264, 279, 282, 297, 300, 315, 318, 333, 336, 351, 354, 369, 372, 387,390, 405, 408, 423, 426, 441, 444, 459, 462, 477, 480, 495, 498, 513,516, 531, 534, 549, 552, 567, 570, 585, 588, 603, 606, 621, or 624.

In another embodiment, the variable domain includes a CDR3 to bereplaced or lacks a CDR3 encoding region and further comprises a CDR1 tobe replaced or lacks a CDR1 encoding region, where the at least onedonor nucleic acid encodes an amino acid sequence substantially as setout in Tables 1 or 7.

In another embodiment, the variable domain includes a CDR3 to bereplaced or lacks a CDR3 encoding region and further comprises a CDR2 tobe replaced or lacks a CDR2 encoding region, where the at least onedonor nucleic acid encodes an amino acid sequence substantially as setout in Tables 1 or 7.

In another embodiment, the variable domain includes a CDR3 to bereplaced or lacks a CDR3 encoding region and further comprises a CDR1and a CDR2 to be replaced or lacks a CDR1 and a CDR2 encoding region,where the at least one donor nucleic acid encodes an amino acid sequencesubstantially as set out in Tables 1 or 7.

Using recombinant DNA methodology, a disclosed CDR sequence may beintroduced into a repertoire of V_(H) or V_(L) domains lacking therespective CDR (Marks et al. (BioTechnology (1992) 10: 779-783). Forexample, a primer adjacent to the 5′ end of the variable domain and aprimer to the third FR can be used to generate a repertoire of variabledomain sequences lacking CDR3. This repertoire can be combined with aCDR3 of a disclosed antibody. Using analogous techniques, portions of adisclosed CDR sequence may be shuffled with portions of CDR sequencesfrom other antibodies to provide a repertoire of antigen-bindingfragments that bind IL-22. Either repertoire can be expressed in a hostsystem such as phage display (described in WO 92/01047 and itscorresponding U.S. Pat. No. 5,969,108) so suitable antigen-bindingfragments that bind to IL-22 can be selected.

A further alternative uses random mutagenesis of the disclosed V_(H) orV_(L) sequences to generate variant V_(H) or V_(L) domains still capableof binding IL-22. A technique using error-prone PCR is described by Gramet al. (Proc. Nat. Acad. Sci. U.S.A. (1992) 89: 3576-3580).

Another method uses direct mutagenesis of the disclosed V_(H) or V_(L)sequences. Such techniques are disclosed by Barbas et al. (Proc. Nat.Acad. Sci. U.S.A. (1994) 91: 3809-3813) and Schier et al. (J. Mol. Biol.(1996) 263: 551-567).

A portion of a variable domain will comprise at least one CDR regionsubstantially as set out herein and, optionally, intervening frameworkregions from the V_(H) or V_(L) domains as set out herein. The portionmay include the C-terminal half of FR1 and/or the N-terminal half ofFR4. Additional residues at the N-terminal or C-terminal end of thevariable domain may not be the same residues found in naturallyoccurring antibodies. For example, construction of antibodies byrecombinant DNA techniques often introduces N- or C-terminal residuesfrom its use of linkers. Some linkers may be used to join variabledomains to other variable domains (e.g., diabodies), constant domains,or proteinaceous labels.

Although the embodiments illustrated in the Examples comprise a“matching” pair of V_(H) and V_(L) domains, a skilled artisan willrecognize that alternative embodiments may comprise antigen-bindingfragments containing only a single CDR from either V_(L) or V_(H)domain. Either one of the single chain specific antigen-binding domainscan be used to screen for complementary domains capable of forming atwo-domain specific antigen-binding fragment capable of, for example,binding to IL-22. The screening may be accomplished by phage displayscreening methods using the so-called hierarchical dual combinatorialapproach disclosed in WO 92/01047. In this approach, an individualcolony containing either a H or L chain clone is used to infect acomplete library of clones encoding the other chain (L or H), and theresulting two-chain specific antigen-binding domain is selected inaccordance with phage display techniques as described.

In some alternative embodiments, the anti-IL-22 antibodies can be linkedto a protein (e.g., albumin) by chemical cross-linking or recombinantmethods. The disclosed antibodies may also be linked to a variety ofnonproteinaceous polymers (e.g., polyethylene glycol, polypropyleneglycol, or polyoxyalkylenes) in manners set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337. Theantibodies can be chemically modified by covalent conjugation to apolymer, for example, to increase their half-life in blood circulation.Exemplary polymers and attachment methods are shown in U.S. Pat. Nos.4,766,106; 4,179,337; 4,495,285; and 4,609,546.

The disclosed antibodies can be modified to alter their glycosylation;that is, at least one carbohydrate moiety can be deleted or added to theantibody. Deletion or addition of glycosylation sites can beaccomplished by changing amino acid sequence to delete or createglycosylation consensus sites, which are well known in the art. Anothermeans of adding carbohydrate moieties is the chemical or enzymaticcoupling of glycosides to amino acid residues of the antibody (see WO87/05330 and Aplin et al. (1981) CRC Crit. Rev. Biochem., 22: 259-306).Removal of carbohydrate moieties can also be accomplished chemically orenzymatically (see Hakimuddin et al. (1987) Arch. Biochem. Biophys.,259: 52; Edge et al. (1981) Anal. Biochem., 118:131; Thotakura et al.(1987) Meth. Enzymol., 138: 350).

Methods for altering an antibody constant region are known in the art.Antibodies with altered function (e.g., altered affinity for an effectorligand such as FcR on a cell or the C1 component of complement) can beproduced by replacing at least one amino acid residue in the constantportion of the antibody with a different residue (see e.g., EP 388,151A1, U.S. Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260). Similar typesof alterations could be described which if applied to a murine or otherspecies antibody would reduce or eliminate similar functions.

For example, it is possible to alter the affinity of an Fc region of anantibody (e.g., an IgG, such as a human IgG) for FcR (e.g., Fc gamma R1)or C1q. The affinity may be altered by replacing at least one specifiedresidue with at least one residue having an appropriate functionality onits side chain, or by introducing a charged functional group, such asglutamate or aspartate, or perhaps an aromatic non-polar residue such asphenylalanine, tyrosine, tryptophan or alanine (see e.g., U.S. Pat. No.5,624,821).

For example, replacing residue 297 (asparagine) with alanine in the IgGconstant region significantly inhibits recruitment of effector cells,while only slightly reducing (about three fold weaker) affinity for Clq(see e.g., U.S. Pat. No. 5,624,821). The numbering of the residues inthe heavy chain is that of the EU index (see Kabat et al., 1991 supra).This alteration destroys the glycosylation site and it is believed thatthe presence of carbohydrate is required for Fc receptor binding. Anyother substitution at this site that destroys the glycosylation site isbelieved to cause a similar decrease in lytic activity. Other amino acidsubstitutions, e.g., changing any one of residues 318 (Glu), 320 (Lys)and 322 (Lys), to Ala, are also known to abolish Clq binding to the Fcregion of IgG antibodies (see e.g., U.S. Pat. No. 5,624,821).

Modified antibodies can be produced which have a reduced interactionwith an Fc receptor. For example, it has been shown that in human IgG₃,which binds to the human Fc gamma R1 receptor, changing Leu 235 to Gludestroys its interaction with the receptor. Mutations on adjacent orclose sites in the hinge link region of an antibody (e.g., replacingresidues 234, 236 or 237 with Ala) can also be used to affect antibodyaffinity for the Fc gamma R1 receptor. The numbering of the residues inthe heavy chain is based in the EU index (see Kabat et al., 1991 supra).

Additional methods for altering the lytic activity of an antibody, forexample, by altering at least one amino acid in the N-terminal region ofthe CH2 domain, are described in WO 94/29351 by Morgan et al. and U.S.Pat. No. 5,624,821.

The antibodies of this invention may be tagged with a detectable orfunctional label. These labels include radiolabels (e.g., ¹³¹I or ⁹⁹Tc),enzymatic labels (e.g., horseradish peroxidase or alkaline phosphatase),and other chemical moieties (e.g., biotin).

The invention may also feature an isolated antibody that binds to IL-22,in particular, human IL-22. In certain embodiments, the anti-IL-22antibody may have at least one of the following characteristics: (1) itis a monoclonal or single specificity antibody; (2) it is a humanantibody; (3) it is an in vitro generated antibody; (4) it is an in vivogenerated antibody (e.g., transgenic mouse system); (5) it binds toIL-22 with an association constant of at least 1012 M⁻¹; (6) it binds toIL-22 with an association constant of at least 10¹¹ M⁻¹; (7) it binds toIL-22 with an association constant of at least 10¹⁰ M⁻¹; (8) it binds toIL-22 with an association constant of at least 10⁹ M⁻¹; (9) it binds toIL-22 with an association constant of at least 10⁶ M⁻¹; (10) it binds toIL-22 with a dissociation constant of 500 nM or less; (11) it binds toIL-22 with a dissociation constant of 10 nM or less; (12) it binds toIL-22 with a dissociation constant of 150 pM or less; (13) it binds toIL-22 with a dissociation constant of 60 pM or less; (14) it inhibitsbinding of IL-22 to IL-22R or a receptor complex of IL-22R and IL-10R2with an IC₅₀ of 10 nM or less; (15) it blocks IL-22 mediatedproliferation of IL-22 receptor engineered BaF3 cells with an IC₅₀ of 1nM or less in one embodiment, with an IC₅₀ of 150 pM or less in anotherembodiment, with an IC₅₀ of 100 pM or less in another embodiment, andwith an IC₅₀ of 10 pM or less in another embodiment; and (16) it blocksIL-22 mediated GROa secretion from HT29 with an IC₅₀ of 1 nM or less inone embodiment, with an IC₅₀ of 150 pM or less in another embodiment,and with an IC₅₀ of 10 pM or less in another embodiment.

One of skill in the art will appreciate that the modifications describedabove are not all-exhaustive, and that many other modifications areobvious to a skilled artisan in light of the teachings of the presentdisclosure.

III. Nucleic Acids, Cloning and Expression Systems

The disclosure provides isolated nucleic acids encoding the disclosedantibodies. The nucleic acids may comprise DNA or RNA, and they may besynthetic (completely or partially) or recombinant (completely orpartially). Reference to a nucleotide sequence as set out hereinencompasses a DNA molecule with the specified sequence, and encompassesa RNA molecule with the specified sequence in which U is substituted forT.

Also provided are nucleic acids that comprise a coding sequence for one,two, or three CDR's, a V_(H) domain, a V_(L) domain, or combinationsthereof, as disclosed herein, or a sequence substantially identicalthereto (e.g., a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% orhigher identical thereto, or which is capable of hybridizing understringent conditions to the sequences disclosed).

In one embodiment, the isolated nucleic acids have nucleotide sequencesencoding heavy chain and light chain variable regions of an anti-IL-22antibody having at least one CDR chosen from the amino acid sequences ofSEQ ID NO: 8-13, 26-31, 44-49, 62-67, 80-85, 98-103, 116-121, 134-139,152-157, 170-175, 188-193, 206-211, 224-229, 242-247, 260-265, 278-283,296-301, 314-319, 332-337, 350-355, 368-373, 386-391, 404-409, 422-427,440-445, 458-463, 476-481, 494-499, 512-517, 530-535, 548-553, 566-571,584-589, 602-607, or 620-625; or sequence encoding a CDR which differsby one or two amino acids from the sequences described herein.

The nucleic acid can encode only the light chain or the heavy chainvariable region, or can also encode an antibody light or heavy chainconstant region, operatively linked to the corresponding variableregion. In one embodiment, the light chain variable region is linked toa constant region chosen from a kappa or a lambda constant region. Thelight chain constant region may also be a human kappa or lambda type. Inanother embodiment, the heavy chain variable region is linked to a heavychain constant region of an antibody isotype chosen from IgG (e.g.,IgG₁, IgG₂, IgG₃, IgG₄), IgM, IgA₁, IgA₂, IgD, and IgE. The heavy chainconstant region may be an IgG (e.g., an IgG₁) isotype.

The nucleic acid compositions of the present invention, while often inthe native sequence (of cDNA or genomic DNA or mixtures thereof) exceptfor modified restriction sites and the like, may be mutated inaccordance with standard techniques to provide gene sequences. Forcoding sequences, these mutations, may affect amino acid sequence asdesired. In particular, nucleotide sequences substantially identical toor derived from native V, D, J, constant, switches and other suchsequences described herein are contemplated (where “derived” indicatesthat a sequence is identical or modified from another sequence).

In one embodiment, the nucleic acid differs (e.g., differs bysubstitution, insertion, or deletion) from that of the sequencesprovided (e.g., as follows: by at least one but less than 10, 20, 30, or40 nucleotides; at least one but less than 1%, 5%, 10% or 20% of thenucleotides in the subject nucleic acid). If necessary for this analysisthe sequences should be aligned for maximum homology. “Looped” outsequences from deletions or insertions, or mismatches, are considereddifferences. The difference may be at a nucleotide(s) encoding anon-essential residue(s), or the difference may be a conservativesubstitution(s).

The disclosure also provides nucleic acid constructs in the form ofplasmids, vectors, transcription or expression cassettes, which compriseat least one nucleic acid as described herein.

The disclosure further provides a host cell that comprises at least onenucleic acid construct described herein.

Also provided are the methods of making the encoded protein(s) from thenucleic acid(s) comprising sequence described herein. The methodcomprises culturing host cells under appropriate conditions so theyexpress the protein from the nucleic acid. Following expression andproduction, the V_(H) or V_(L) domain, or specific binding member may beisolated and/or purified using any suitable technique, then used asappropriate. The method can also include the steps of fusing a nucleicacid encoding a scF_(v) with nucleic acids encoding a Fc portion of anantibody and expressing the fused nucleic acid in a cell. The method canalso include a step of germlining.

Antigen-binding fragments, V_(H) and/or V_(L) domains, and encodingnucleic acid molecules and vectors may be isolated and/or purified fromtheir natural environment, in substantially pure or homogenous form, or,in the case of nucleic acid, free or substantially free of nucleic acidor genes of origin other than the sequence encoding a polypeptide withthe require function.

Systems for cloning and expressing polypeptides in a variety of hostcells are known in the art. Cells suitable for producing antibodies aredescribed in, for example, Fernandez et al. (1999) Gene ExpressionSystems, Academic Press, eds. In brief, suitable host cells includemammalian cells, insect cells, plant cells, yeast cells, or prokaryoticcells, e.g., E. coli. Mammalian cells available in the art forheterologous polypeptide expression include lymphocytic cell lines(e.g., NSO), HEK293 cells, Chinese hamster ovary (CHO) cells, COS cells,HeLa cells, baby hamster kidney cells, oocyte cells, and cells from atransgenic animal, e.g., mammary epithelial cell. In one embodiment, theGIL01, GIL16, GIL45, GIL60, GIL68, GIL92, 097D09, 062A09, 062G05,087B03, 367D04, 368D04, 166B06, 166G05, 375G06, 376B10, 354A08, 355B06,355E04, and 356A11 antibodies are expressed in HEK293 or CHO cells. Inanother embodiment, a selection of antibodies chosen from 365A11,354A08, 087B03, and 368D04 are expressed in HEK293 or CHO cells. Inother embodiments, the nucleic acids encoding the antibodies of theinvention are placed under the control of a tissue-specific promoter(e.g., a mammary specific promoter) and the antibodies are produced intransgenic animals. For example, the antibodies are secreted into themilk of the transgenic animal, such as a transgenic cow, pig, horse,sheep, goat or rodent.

Suitable vectors may be chosen or constructed to contain appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genes,and other sequences. The vectors may also contain a plasmid or viralbackbone. For details, see Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press (1989).Many established techniques used with vectors, including themanipulation, preparation, mutagenesis, sequencing, and transfection ofDNA, are described in Current Protocols in Molecular Biology, SecondEdition, Ausubel et al. eds., John Wiley & Sons (1992).

A further aspect of the disclosure provides a method of introducing thenucleic acid into a host cell. For eukaryotic cells, suitabletransfection techniques may include calcium phosphate, DEAE-Dextran,electroporation, liposome-mediated transfection, and transduction usingretrovirus or other viruses, e.g., vaccinia or baculovirus. Forbacterial cells, suitable techniques may include calcium chloridetransformation, electroporation, and transfection using bacteriophage.DNA introduction may be followed by a selection method (e.g., drugresistance) to select cells that contain the nucleic acid.

IV. Uses of Anti-IL-22 Antibodies

Anti-IL-22 antibodies that act as antagonists to IL-22 can be used toregulate at least one IL-22-mediated immune response, such as acting onepithelial cells in solid tissue and indirectly modulating downstreamimmune responses, such as blocking expansion of T cell subsets,including, for example, T_(H)17 T cells. In one embodiment, antibodiesof the invention are used in a method for regulating an immune response,the method comprising contacting IL-22 with an antibody of the inventionthereby regulating the immune response. In one embodiment, the immuneresponse comprises cell proliferation, cytolytic activity, cytokinesecretion, or chemokine secretion.

Accordingly, the antibodies of the invention can be used to directly orindirectly inhibit the activity (e.g., proliferation, differentiation,and/or survival) of an immune or hematopoietic cell (e.g., a cell ofmyeloid, lymphoid, or erythroid lineage, or precursor cells thereof),and, thus, can be used to treat a variety of immune disorders andhyperproliferative disorders. Non-limiting examples of immune disordersthat can be treated include, but are not limited to, autoimmunedisorders, e.g., arthritis (including rheumatoid arthritis, juvenilerheumatoid arthritis, osteoarthritis, psoriatic arthritis,lupus-associated arthritis or ankylosing spondylitis), scleroderma,systemic lupus erythematosis, HIV, Sjogren's syndrome, vasculitis,multiple sclerosis, autoimmune thyroiditis, dermatitis (including atopicdermatitis and eczematous dermatitis), myasthenia gravis, inflammatorybowel disease (IBD), Crohn's disease, colitis, diabetes mellitus (typeI); inflammatory conditions of, e.g., the skin (e.g., psoriasis),cardiovascular system (e.g., atherosclerosis), nervous system (e.g.,Alzheimer's disease), liver (e.g., hepatitis), kidney (e.g., nephritis)and pancreas (e.g., pancreatitis); cardiovascular disorders, e.g.,cholesterol metabolic disorders, oxygen free radical injury, ischemia;disorders associated with wound healing; respiratory disorders, e.g.,asthma and COPD (e.g., cystic fibrosis); acute inflammatory conditions(e.g., endotoxemia, sepsis and septicaemia, toxic shock syndrome andinfectious disease); transplant rejection and allergy. In oneembodiment, the IL-22-associated disorder is, an arthritic disorder,e.g., a disorder chosen from one or more of rheumatoid arthritis,juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, orankylosing spondylitis; a respiratory disorder (e.g., asthma, chronicobstructive pulmonary disease (COPD); or an inflammatory condition of,e.g., the skin (e.g., psoriasis), cardiovascular system (e.g.,atherosclerosis), nervous system (e.g., Alzheimer's disease), liver(e.g., hepatitis), kidney (e.g., nephritis), pancreas (e.g.,pancreatitis), and gastrointestinal organs, e.g., colitis, Crohn'sdisease and IBD; acute inflammatory conditions, e.g., endotoxemia,sepsis and septicaemia, toxic shock syndrome and infectious disease;multiple organ failure; respiratory disease (ARD); amyloidosis;nephropathies such as glomerulosclerosis, membranous neuropathy, renalarteriosclerosis, glomerulonephritis, fibroproliferative diseases of thekidney, as well as other kidney disfunctions and renal tumors. Becauseof IL-22's effects on epithelia, anti-IL-22 antibodies can be used totreat epithelial cancers, e.g., carcinoma, melanoma and others. For adescription of a rationale for IL-22 inhibition in these and otherdisease states see WO 03/083062 (pages 58-75).

Multiple sclerosis is a central nervous system disease that ischaracterized by inflammation and loss of myelin sheaths—the fattymaterial that insulates nerves and is needed for proper nerve function.Inflammation that results from an immune response that is dependent onIL-22 can be treated with the antibodies and compositions of thisinvention. In the experimental autoimmune encephalitis (EAE) mouse modelfor multiple sclerosis (Tuohy et al. (J. Immunol. (1988) 141:1126-1130), Sobel et al. (J. Immunol. (1984) 132: 2393-2401), andTraugott (Cell Immunol. (1989) 119: 114-129), treatment of mice withGIL01, GIL16, GIL45, GIL60, GIL68, GIL92, 097D09, 062A09, 062G05,087B03, 367D04, 368D04, 166B06, 166G05, 375G06, 376B10, 354A08, 355B06,355E04, or 356A11 injections prior (and continuously) to EAE inductionmay profoundly delay the onset of the disease. This can serve as a modelfor confirming use of the antibody of the invention. The antibodies ofthis invention may similarly be used to treat multiple sclerosis inhumans.

Arthritis is a disease characterized by inflammation in the joints.Rheumatoid Arthritis (RA) is the most frequent form of arthritis,involving inflammation of connective tissue and the synovial membrane, amembrane that lines the joint. The inflamed synovial membrane ofteninfiltrates the joint and damages joint cartilage and bone. IL-22 andIL-22R protein and/or transcript is associated with both human diseases.In RA synovial biopsies, IL-22 protein is detected in vimentin⁺ synovialfibroblasts and some CD68⁺ macrophages while IL-22R is detected insynovial fibroblasts. Treatment of synovial fibroblasts with IL-22induces the production of monocyte chemoattractant protein-1, MCP-1, aswell as general metabolic activity (Ikeuchi, H., et al. (2005) ArthritisRheum. 52:1037-46). Inhibitors of IL-22 ameliorate symptoms ofrheumatoid arthritis (WO 2005/000897 A2; U.S. Pat. No. 6,939,545).Increased secretion of inflammatory cytokines and chemokines, and moreimportantly, increased disease resulting from immune responses that aredependent on IL-22 may be treated with the antibodies of this invention.Similarly, the antibodies and compositions of this invention may be usedto treat RA or other arthritic diseases in humans.

Transplant rejection is the immunological phenomenon where tissues froma donor are specifically “attacked” by immune cells of the host. Theprinciple “attacking” cells are T cells, whose T cell receptorsrecognize the donor's MHC molecules as “foreign.” This recognitionactivates the T cells, which proliferate and secrete a variety ofcytokines and cytolytic proteins that ultimately destroy the transplant.MLR and transplantation models have been described by Current Protocolsin Immunology, Second Edition, Coligan et al. eds., John Wiley & Sons,1994; Kasaian et al. (Immunity (2002) 16: 559-569); Fulmer et al. (Am.J. Anat. (1963) 113: 273-285), and Lenschow et al. (Science (1992) 257:789-792). The antibodies and compositions of this invention may be usedto reduce the MLR and treat transplant rejection and related diseases(e.g., graft versus host disease) in humans that are dependent on IL-22.

The antibodies of this invention can also be used to treathyperproliferative disorders associated with aberrant activity ofIL-22-responsive cells and IL-22R/IL-10R2-responsive cells byadministering the antibodies in an amount sufficient to inhibit orreduce hyperproliferation of IL-22 and/or IL-22R and/orIL-10R2-responsive cells in a subject and allowing the antibodies totreat or prevent the disorder. IL-22 and IL-22R expression isconstitutive on epithelial cells in a number of tissues including, butnot limited to, pancreas, lung, skin, gut, liver, kidney (Kotenko, S. V.et al. (2001) J. Biol. Chem. 276:2725-32; Xie, M. H. et al. (2000) J.Biol. Chem. 275:31335-9; Wolk, K. et al. (2004) Immunity 21:241-54). Inaddition, IL-22 receptor complex is also expressed on the surface offibroblasts from the diseased joint and normal gut (Ikeuchi, H. et al.(2005) Arthritis Rheum. 52:1037-46; Andoh, A. et al. (2005)Gastroenterology 129:969-84). Neoplastic derivatives of these cell typesmay be hyper responsive to IL-22, modulating these cells ability tosurvive in the organism. Hence antibodies to IL-22 may be used toinhibit the progression of such neoplasms, e.g. squamous cellcarcinomas, basal cell carcinomas, transitional cell papillomas andcarcinomas, adenomas, adenocarcinoma, linitis plastica, insulinoma,glucagonoma, gastrinoma, vipoma, cholangiocarcinoma, hepatocellularcarcinoma, adenoid cyctic carcinoma, carcinoid tumor of appendix,prolactinoma, oncocytoma, hurthle cell adenoma, renal cell carcinoma,Grawitz tumor, multiple endocrine adenomas, endometroid adenoma, adnexaland skin appendage neoplasms, mucoepidermoid neoplasms, cystic, mucinousand serous neoplasms, cystadenoma, pseudomyxoma peritonei, ductal,lobular and medullary neoplasms, acinar cell neoplasms, complexepithelial neoplasms, Warthin's tumor, thymoma, specialized gonadalneoplasms, sex cord-stromal tumor, thecoma, granulosa cell tumor,arrhenoblastoma, sertoli-leydig cell tumor, paraganglioma,pheochromocytoma, glomus tumor, malanocytic nevus, malignant melanoma,melanoma, nodular melanoma, dysplastic nevus, lentigo maligna,superficial spreading melanoma, or acral lentiginous melanoma. While theIL-22 receptor is not detected on ex vivo naïve or activated immunecells, dysregulation of the receptor might make such derivativeneoplastic cells responsive to IL-22 and thus inhibition by an antibodyto IL-22.

In another aspect, the invention features a method of decreasing,inhibiting or reducing an acute phase response in a subject. The methodincludes administering to the subject an anti-IL-22 antibody or fragmentthereof as described herein, in an amount sufficient to decrease,inhibit or reduce the acute phase response in the subject. In oneembodiment, the subject is a mammal, e.g., a human suffering from anIL-22-associated disorder as described herein, including, e.g.,respiratory disorders, inflammatory disorders and autoimmune disorders.In one embodiment, the IL-22 binding agent is administered locally,e.g., topically, subcutaneously, or other administrations that are notin the general circulation.

IL-22 is believed to exert its inflammatory effects locally, e.g. byacting (e.g., directly acting) as a modular or a regulator of tissueinflammation as opposed to direct systemic effects. Accordingly,inhibition of IL-22 activity using, e.g. an anti-IL-22 antibody of thepresent invention may provide a more effective (e.g., less toxic)tissue-specific, anti-inflammatory agent than systemic anti-inflammatorymodalities. Furthermore, inhibition of local IL-22 using, e.g., ananti-IL-22 antibody or fragment thereof described herein, may provide auseful candidate for combination with systemic anti-inflammatorymodalities.

V. Combination Therapy

In one embodiment, a pharmaceutical composition comprising at least oneanti-IL-22 antibody and at least one therapeutic agent is administeredin combination therapy. The therapy is useful for treating pathologicalconditions or disorders, such as immune and inflammatory disorders. Theterm “in combination” in this context means that the antibodycomposition and the therapeutic agent are given substantiallycontemporaneously, either simultaneously or sequentially. In oneembodiment, if given sequentially, at the onset of administration of thesecond compound, the first of the two compounds is still detectable ateffective concentrations at the site of treatment. In anotherembodiment, if given sequentially, at the onset of administration of thesecond compound, the first of the two compounds is not detectable ateffective concentrations at the site of treatment.

For example, the combination therapy can include at least one anti-IL-22antibody co-formulated with, and/or co-administered with, at least oneadditional therapeutic agent. The additional agents may include at leastone cytokine inhibitor, growth factor inhibitor, immunosuppressant,anti-inflammatory agent, metabolic inhibitor, enzyme inhibitor,cytotoxic agent, and cytostatic agent, as described in more detailbelow. In one embodiment, the additional agent is a standard treatmentfor arthritis, including, but not limited to, non-steroidalanti-inflammatory agents (NSAIDs); corticosteroids, includingprednisolone, prednisone, cortisone, and triamcinolone; and diseasemodifying anti-rheumatic drugs (DMARDs), such as methotrexate,hydroxychloroquine (Plaquenil) and sulfasalazine, leflunomide (Arava),tumor necrosis factor inhibitors, including etanercept (Enbrel),infliximab (Remicade) (with or without methotrexate), and adalimumab(Humira), anti-CD20 antibody (e.g., Rituxan), soluble interleukin-1receptor, such as anakinra (Kineret), gold, minocycline (Minocin),penicillamine, and cytotoxic agents, including azathioprine,cyclophosphamide, and cyclosporine. Such combination therapies mayadvantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible toxicities or complications associatedwith the various monotherapies. Moreover, the additional therapeuticagents disclosed herein act on pathways in addition to or that differfrom the IL-22/IL-22R/IL-10R2 pathway, and thus are expected to enhanceand/or synergize with the effects of the anti-IL-22 antibodies.

Therapeutic agents used in combination with anti-IL-22 antibodies may bethose agents that interfere at different stages in the autoimmune andsubsequent inflammatory response. In one embodiment, at least oneanti-IL-22 antibody described herein may be co-formulated with, and/orco-administered with, at least one cytokine and/or growth factorantagonist. The antagonists may include soluble receptors, peptideinhibitors, small molecules, ligand fusions, antibodies and bindingfragments thereof (that bind cytokines or growth factors or theirreceptors or other cell surface molecules), and “anti-inflammatorycytokines” and agonists thereof.

Non-limiting examples of the agents that can be used in combination withthe anti-IL-22 antibodies described herein, include, but are not limitedto, antagonists of at least one interleukin (e.g., IL-1, IL-2, IL-6,IL-7, IL-8, IL-12 (or one of its subunits p35 or p40), IL-13, IL-15,IL-16, IL-17A-F (including heterodimers thereof, for example,IL-17A/IL-17F heterodimer), IL-18, IL-19, IL-20, IL-21, and IL-23 (orone of its subunits p19 or p40); cytokine (e.g., TNFα, LT, EMAP-II, andGM-CSF); and growth factor (e.g., FGF and PDGF). The agents may alsoinclude, but not limited to, antagonists of at least one receptor for aninterleukin, cytokine, and growth factor. Anti-IL-22 antibodies can alsobe combined with inhibitors (e.g., antibodies or binding fragmentsthereof) to cell surface molecules such as CD2, CD3, CD4, CD8, CD20(e.g. Rituxan), CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86(B7.2), CD90, or their ligands (e.g., CD154 (gp39, CD40L)), orLFA-1/ICAM-1 and VLA-4/VCAM-1 (Yusuf-Makagiansar et al. (2002) Med ResRev 22(2):146-67)). In certain embodiments, antagonists that can be usedin combination with anti-IL-22 antibodies described herein may includeantagonists of IL-1, IL-12 (or one of its subunits p35 or p40), TNFα,IL-15, IL-17A-F (including heterodimers thereof, for example,IL-17A/IL-17F heterodimer), IL-18, IL-19, IL-20, IL-21, and IL-23 (orone of its subunits p19 or p40), and their receptors.

Examples of those agents include IL-12 antagonists (such as antibodiesthat bind IL-12 (see e.g., WO 00/56772) or one of its subunits p35 orp40); IL-12 receptor inhibitors (such as antibodies to the IL-12receptor); and soluble IL-12 receptor and fragments thereof. Examples ofIL-15 antagonists include antibodies against IL-15 or its receptor,soluble fragments of the IL-15 receptor, and IL-15-binding proteins.Examples of IL-18 antagonists include antibodies to IL-18, solublefragments of the IL-18 receptor, and IL-18 binding proteins (IL-18BP,Mallet et al. (2001) Circ. Res. 28). Examples of IL-1 antagonistsinclude Interleukin-1-Converting Enzyme (ICE) inhibitors (such asVx740), IL-1 antagonists (e.g., IL-1RA (ANIKINRA (or Kineret), AMGEN)),sIL-1R11 (Immunex), and anti-IL-1 receptor antibodies.

In one embodiment, the combination therapy includes at least oneanti-IL-22 antibody co-formulated with, and/or co-administered with anantagonist, such as an antibody or antigen binding fragment thereof or asoluble receptor, of at least one of IL-17A, IL-17F, IL-17A/IL-17Fheterodimer, or IL-23 (or one of its subunits p19 or p40).

Examples of TNF antagonists include antibodies to TNF (e.g., humanTNFα), such as D2E7 (human anti-TNFα antibody, U.S. Pat. No. 6,258,562,Humira™, BASF); CDP-571/CDP-870/BAY-10-3356 (humanized anti-TNFαantibodies, Celltech/Pharmacia); cA2 (chimeric anti-TNFα antibody,Remicade™, Centocor); and anti-TNF antibody fragments (e.g., CPD870).Other examples include soluble TNF receptor (e.g., human p55 or p75)fragments and derivatives, such as p55 kdTNFR-IgG (55 kD TNFreceptor-IgG fusion protein, Lenercept™) and 75 kdTNFR-IgG (75 kD TNFreceptor-IgG fusion protein, Enbrel™, Immunex, see, e.g., Arthritis &Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A).Further examples include enzyme antagonists (e.g., TNFα convertingenzyme inhibitors (TACE) such as alpha-sulfonyl hydroxamic acidderivative (WO 01/55112) or N-hydroxyformamide inhibitor (GW 3333, -005,or -022)) and TNF-bp/s-TNFR (soluble TNF binding protein, see e.g.,Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S284; and Am.J. Physiol. Heart Circ. Physiol. (1995) Vol. 268, pp. 37-42). TNFantagonists may be soluble TNF receptor (e.g., human p55 or p75)fragments and derivatives, such as 75 kdTNFR-IgG; and TNFα convertingenzyme (TACE) inhibitors.

In other embodiments, the anti-IL-22 antibodies described herein can beadministered in combination with at least one of the following: IL-13antagonists, such as soluble IL-13 receptors and/or anti-IL-13antibodies; and IL-2 antagonists, such as IL-2 fusion proteins (e.g.,DAB 486-IL-2 and/or DAB 389-IL-2, Seragen, see e.g., Arthritis &Rheumatism (1993) Vol. 36, 1223) and anti-IL-2R antibodies (e.g.,anti-Tac (humanized antibody, Protein Design Labs, see Cancer Res. 1990Mar. 1; 50(5):1495-502)). Another combination includes anti-IL-22antibodies in combination with non-depleting anti-CD4 inhibitors such asIDEC-CE9.1/SB 210396 (anti-CD4 antibody, IDEC/SmithKline). Yet othercombinations include anti-IL-22 antibodies with antagonists (such asantibodies, soluble receptors, or antagonistic ligands) of costimulatorymolecules, such as CD80 (B7.1) and CD86 (B7.2); ICOSL, ICOS, CD28, andCTLA4 (e.g., CTLA4-Ig (atabacept)); P-selectin glycoprotein ligand(PSGL); and anti-inflammatory cytokines and agonists thereof (e.g.,antibodies). The anti-inflammatory cytokines may include IL-4(DNAX/Schering); IL-10 (SCH 52000, recombinant IL-10, DNAX/Schering);IL-13; and TGF.

In other embodiments, at least one anti-IL-22 antibody can beco-formulated with, and/or co-administered with, at least oneanti-inflammatory drug, immunosuppressant, metabolic inhibitor, andenzymatic inhibitor. Non-limiting examples of the drugs or inhibitorsthat can be used in combination with the IL-22 antagonists describedherein, include, but are not limited to, at least one of: non-steroidalanti-inflammatory drug (NSAID) (such as ibuprofen, Tenidap (see e.g.,Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S280)),Naproxen (see e.g., Neuro Report (1996) Vol. 7, pp. 1209-1213),Meloxicam, Piroxicam, Diclofenac, and Indomethacin); Sulfasalazine (seee.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S281);corticosteroid (such as prednisolone); cytokine suppressiveanti-inflammatory drug (CSAID); and an inhibitor of nucleotidebiosynthesis (such as an inhibitor of purine biosynthesis (e.g., folateantagonist such as methotrexate) and an inhibitor of pyrimidinebiosynthesis (e.g., a dihydroorotate dehydrogenase (DHODH) inhibitorsuch as leflunomide (see e.g., Arthritis & Rheumatism (1996) Vol. 39,No. 9 (supplement), S131; Inflammation Research (1996) Vol. 45, pp.103-107)). Therapeutic agents for use in combination with IL-22/IL-22Ror IL-22/IL-10R2 antagonists may include NSAIDs, CSAIDs, DHODHinhibitors (such as leflunomide), and folate antagonists (such asmethotrexate).

Examples of additional inhibitors include at least one of:corticosteroid (oral, inhaled and local injection); immunosuppressant(such as cyclosporin and tacrolimus (FK-506)); a mTOR inhibitor (such assirolimus (rapamycin) or a rapamycin derivative (e.g., ester rapamycinderivative such as CCI-779 (Elit. L. (2002) Current Opinion Investig.Drugs 3(8):1249-53; Huang, S. et al. (2002) Current Opinion Investig.Drugs 3(2):295-304))); an agent which interferes with the signaling ofproinflammatory cytokines such as TNFα and IL-1 (e.g., IRAK, NIK, IKK,p38 or a MAP kinase inhibitor); a COX2 inhibitor (e.g., celecoxib andvariants thereof (MK-966), see e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (supplement), S81); a phosphodiesterase inhibitor (such asR973401, see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S282)); a phospholipase inhibitor (e.g., an inhibitor ofcytosolic phospholipase 2 (cPLA2) such as trifluoromethyl ketone analogs(U.S. Pat. No. 6,350,892)); an inhibitor of vascular endothelial cellgrowth factor (VEGF); an inhibitor of the VEGF receptor; and aninhibitor of angiogenesis. Therapeutic agents for use in combinationwith anti-IL-22 antibodies may include immunosuppresants (such ascyclosporine and tacrolimus (FK-506)); and mTOR inhibitors (such assirolimus (rapamycin) or rapamycin derivatives (e.g., ester rapamycinderivatives such as CCI-779)); COX2 inhibitors (such as celecoxib andvariants thereof); and phospholipase inhibitors (such as inhibitors ofcytosolic phospholipase 2 (cPLA2) (e.g., trifluoromethyl ketoneanalogs)).

Examples of therapeutic agents that can be co-administered and/orco-formulated with at least one anti-IL-22 antibody, include, but arenot limited to, at least one of: TNF antagonists (such as anti-TNFantibodies); soluble fragments of TNF receptors (e.g., human p55 andp75) and derivatives thereof (such as p55 kdTNFR-IgG (55 kD TNFreceptor-IgG fusion protein, Lenercept™) and 75 kdTNFR-IgG (75 kD TNFreceptor-IgG fusion protein, Enbrel™)); TNF enzyme antagonists (such asTACE inhibitors); antagonists of IL-12 (or one of its subunits p35 orp40), IL-15, IL-17A-F (including heterodimers thereof, for example,IL-17A/IL-17F heterodimer), IL-18, IL-19, IL-20, IL-21, IL-22, and IL-23(or one of its subunits p19 or p40); T cell and B cell depleting agents(such as anti-CD4 or anti-CD22 antibodies); small molecule inhibitors(such as methotrexate and leflunomide); sirolimus (rapamycin) andanalogs thereof (such as CCI-779); Cox-2 and cPLA2 inhibitors; p38,TPL-2, Mk-2 and NFκB inhibitors; RAGE and soluble RAGE; P-selectin andPSGL-1 inhibitors (such as antibodies to and small molecule inhibitors);and estrogen receptor beta (ERB) agonists, and ERB-NFkb antagonists.Therapeutic agents that can be co-administered and/or co-formulated withat least one anti-IL-22 antibody may include at least one of: a solublefragment of a TNF receptor (e.g., human p55 or p75) such as 75kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein, Enbrel™);methotrexate; leflunomide; and sirolimus (rapamycin) and analogs thereof(such as CCI-779).

The anti-IL-22 antibodies disclosed herein can be used in combinationwith other therapeutic agents to treat specific immune disorders asdiscussed in further detail below.

Non-limiting examples of agents for treating arthritic disorders (e.g.,rheumatoid arthritis, inflammatory arthritis, rheumatoid arthritis,juvenile rheumatoid arthritis, osteoarthritis and psoriatic arthritis),with which an anti-IL-22 antibody can be combined include at least oneof the following: TNF antagonists (such as anti-TNF antibodies); solublefragments of TNF receptors (e.g., human p55 and p75) and derivativesthereof (such as p55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein,Lenercept™) and 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein,Enbrel™)); TNF enzyme antagonists (such as TACE inhibitors); antagonistsof IL-12 (or one of its subunits p35 or p40), IL-15, IL-17A-F (includingheterodimers thereof, for example, IL-17A/IL-17F heterodimer), IL-18,IL-19, IL-20, IL-21, IL-23 (or one of its subunits p19 or p40), andIL-24; T cell and B cell depleting agents (such as anti-CD4, anti-CD20,or anti-CD22 antibodies); small molecule inhibitors (such asmethotrexate and leflunomide); sirolimus (rapamycin) and analogs thereof(e.g., CCI-779); Cox-2 and cPLA2 inhibitors; NSAIDs; p38, TPL-2, Mk-2,and NFκB inhibitors; RAGE or soluble RAGE; P-selectin or PSGL-1inhibitors (such as small molecule inhibitors and antibodies to);estrogen receptor beta (ERB) agonists, and ERB-NFκB antagonists.Therapeutic agents that can be co-administered and/or co-formulated withat least one IL-22/IL-22R/IL-10R2 antagonist may include at least oneof: a soluble fragment of a TNF receptor (e.g., human p55 or p75) suchas 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein, Enbrel™);methotrexate; leflunomide; and sirolimus (rapamycin) or an analogthereof (e.g., CCI-779).

Non-limiting examples of agents for treating multiple sclerosis withwhich anti-IL-22 antibody can be combined include interferon-β forexample, IFNβ-1a and IFNβ-1b), copaxone, corticosteroids, IL-1inhibitors, TNF inhibitors, antibodies to CD40 ligand, antibodies toCD80, and IL-12 antagonists, including antibodies that bind IL-12 (orone of its subunits p35 or p40).

Non-limiting examples of agents for treating inflammatory bowel diseaseor Crohn's disease with which an anti-IL-22 antibody can be combinedinclude budenoside; epidermal growth factor; corticosteroids;cyclosporine; sulfasalazine; aminosalicylates; 6-mercaptopurine;azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine;olsalazine; balsalazide; antioxidants; thromboxane inhibitors; IL-1receptor antagonists; anti-IL-1 monoclonal antibodies; anti-IL-6monoclonal antibodies; growth factors; elastase inhibitors;pyridinyl-imidazole compounds; TNF antagonists as described herein;IL-4, IL-10, IL-13, and/or TGFβ or agonists thereof (e.g., agonistantibodies); IL-11; glucuronide- or dextran-conjugated prodrugs ofprednisolone, dexamethasone or budesonide; ICAM-1 antisensephosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals,Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.);slow-release mesalazine; methotrexate; antagonists of PlateletActivating Factor (PAF); ciprofloxacin; and lignocaine.

In other embodiments, an anti-IL-22 antibody can be used in combinationwith at least one antibody directed at other targets involved inregulating immune responses, e.g., transplant rejection or graft versushost disease. Non-limiting examples of agents for treating immuneresponses with which an IL-22/IL-22R/IL10R2 antagonist of the inventioncan be combined include the following: antibodies against cell surfacemolecules, including but not limited to CD25 (IL-2 receptor α), CD11a(LFA-1), CD54 (ICAM-1), CD4, CD45, CD28/CTLA4, CD80 (B7-1), CD86 (B7-2),or combinations thereof. In another embodiment, an anti-IL-22 antibodyis used in combination with at least one general immunosuppressiveagent, such as cyclosporin A or FK506.

Another aspect of the present invention accordingly relates to kits forcarrying out the combined administration of the anti-IL-22 antibodieswith other therapeutic agents. In one embodiment, the kit comprises atleast one anti-IL-22 antibody formulated in a pharmaceutical carrier,and at least one therapeutic agent, formulated as appropriate in one ormore separate pharmaceutical preparations.

VI. Diagnostic Uses

The antibodies may also be used to detect the presence of IL-22 inbiological samples. By correlating the presence or level of theseproteins with a medical condition, one of skill in the art can diagnosethe associated medical condition. For example, IL-22 induces changesassociated with those caused by inflammatory cytokines (such as IL-1 andTNFα), and inhibitors of IL-22 ameliorate symptoms of rheumatoidarthritis (WO 2005/000897 A2). Illustrative medical conditions that maybe diagnosed by the antibodies of this invention include multiplesclerosis, rheumatoid arthritis, psoriasis, inflammatory bowel disease,pancreatitis, and transplant rejection.

Antibody-based detection methods are well known in the art, and includeELISA, radioimmunoassays, immunoblots, Western blots, flow cytometry,immunofluorescence, immunoprecipitation, and other related techniques.The antibodies may be provided in a diagnostic kit that incorporates atleast one of these procedures to detect IL-22. The kit may contain othercomponents, packaging, instructions, or other material to aid thedetection of the protein and use of the kit.

Antibodies may be modified with detectable markers, including ligandgroups (e.g., biotin), fluorophores and chromophores, radioisotopes,electron-dense reagents, or enzymes. Enzymes are detected by theiractivity. For example, horseradish peroxidase is detected by its abilityto convert tetramethylbenzidine (TMB) to a blue pigment, quantifiablewith a spectrophotometer. Other suitable binding partners include biotinand avidin, IgG and protein A, and other receptor-ligand pairs known inthe art.

Antibodies can also be functionally linked (e.g., by chemical coupling,genetic fusion, non-covalent association or otherwise) to at least oneother molecular entity, such as another antibody (e.g., a bispecific ora multispecific antibody), toxins, radioisotopes, cytotoxic orcytostatic agents, among others. Other permutations and possibilitiesare apparent to those of ordinary skill in the art, and they areconsidered equivalents within the scope of this invention.

VII. Pharmaceutical Compositions and Methods of Administration

Certain embodiments of the invention include compositions comprising thedisclosed antibodies. The compositions may be suitable forpharmaceutical use and administration to patients. The compositionscomprise an antibody of the present invention and a pharmaceuticalexcipient. As used herein, “pharmaceutical excipient” includes solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, etc., that are compatible withpharmaceutical administration. Use of these agents for pharmaceuticallyactive substances is well known in the art. The compositions may alsocontain other active compounds providing supplemental, additional, orenhanced therapeutic functions. The pharmaceutical compositions may alsobe included in a container, pack, or dispenser together withinstructions for administration.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Methods toaccomplish the administration are known to those of ordinary skill inthe art. Pharmaceutical compositions may be topically or orallyadministered, or capable of transmission across mucous membranes.Examples of administration of a pharmaceutical composition include oralingestion or inhalation. Administration may also be intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, cutaneous, ortransdermal.

Solutions or suspensions used for intradermal or subcutaneousapplication typically include at least one of the following components:a sterile diluent such as water, saline solution, fixed oils,polyethylene glycol, glycerine, propylene glycol, or other syntheticsolvent; antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate,citrate, or phosphate; and tonicity agents such as sodium chloride ordextrose. The pH can be adjusted with acids or bases. Such preparationsmay be enclosed in ampoules, disposable syringes, or multiple dosevials.

Solutions or suspensions used for intravenous administration include acarrier such as physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.), ethanol, or polyol. In all cases, thecomposition must be sterile and fluid for easy syringability. Properfluidity can often be obtained using lecithin or surfactants. Thecomposition must also be stable under the conditions of manufacture andstorage. Prevention of microorganisms can be achieved with antibacterialand antifungal agents, e.g., parabens, chlorobutanol, phenol, ascorbicacid, thimerosal, etc. In many cases, isotonic agents (sugar),polyalcohols (mannitol and sorbitol), or sodium chloride may be includedin the composition. Prolonged absorption of the composition can beaccomplished by adding an agent which delays absorption, e.g., aluminummonostearate and gelatin.

Oral compositions include an inert diluent or edible carrier. Thecomposition can be enclosed in gelatin or compressed into tablets. Forthe purpose of oral administration, the antibodies can be incorporatedwith excipients and placed in tablets, troches, or capsules.Pharmaceutically compatible binding agents or adjuvant materials can beincluded in the composition. The tablets, troches, and capsules, maycontain (1) a binder such as microcrystalline cellulose, gum tragacanthor gelatin; (2) an excipient such as starch or lactose, (3) adisintegrating agent such as alginic acid, Primogel, or corn starch; (4)a lubricant such as magnesium stearate; (5) a glidant such as colloidalsilicon dioxide; or (6) a sweetening agent or a flavoring agent.

The composition may also be administered by a transmucosal ortransdermal route. For example, antibodies that comprise a Fc portionmay be capable of crossing mucous membranes in the intestine, mouth, orlungs (via Fc receptors). Transmucosal administration can beaccomplished through the use of lozenges, nasal sprays, inhalers, orsuppositories. Transdermal administration can also be accomplishedthrough the use of a composition containing ointments, salves, gels, orcreams known in the art. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used. Foradministration by inhalation, the antibodies are delivered in an aerosolspray from a pressured container or dispenser, which contains apropellant (e.g., liquid or gas) or a nebulizer.

In certain embodiments, the antibodies of this invention are preparedwith carriers to protect the antibodies against rapid elimination fromthe body. Biodegradable polymers (e.g., ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylacticacid) are often used. Methods for the preparation of such formulationsare known by those skilled in the art. Liposomal suspensions can be usedas pharmaceutically acceptable carriers too. The liposomes can beprepared according to established methods known in the art (U.S. Pat.No. 4,522,811).

The antibodies or antibody compositions of the invention areadministered in therapeutically effective amounts as described.Therapeutically effective amounts may vary with the subject's age,condition, sex, and severity of medical condition. Appropriate dosagemay be determined by a physician based on clinical indications. Theantibodies or compositions may be given as a bolus dose to maximize thecirculating levels of antibodies for the greatest length of time.Continuous infusion may also be used after the bolus dose.

As used herein, the term “subject” is intended to include human andnon-human animals. Subjects may include a human patient having adisorder characterized by cells that express IL-22, e.g., a cancer cellor an immune cell. The term “non-human animals” of the inventionincludes all vertebrates, such as non-human primates, sheep, dogs, cows,chickens, amphibians, reptiles, etc.

Examples of dosage ranges that can be administered to a subject can bechosen from: 1 μg/kg to 20 mg/kg, 1 μg/kg to 10 mg/kg, 1 μg/kg to 1mg/kg, 10 μg/kg to 1 mg/kg, 10 μg/kg to 100 μg/kg, 100 μg/kg to 1 mg/kg,250 μg/kg to 2 mg/kg, 250 μg/kg to 1 mg/kg, 500 μg/kg to 2 mg/kg, 500μg/kg to 1 mg/kg, 1 mg/kg to 2 mg/kg, 1 mg/kg to 5 mg/kg, 5 mg/kg to 10mg/kg, 10 mg/kg to 20 mg/kg, 15 mg/kg to 20 mg/kg, 10 mg/kg to 25 mg/kg,15 mg/kg to 25 mg/kg, 20 mg/kg to 25 mg/kg, and mg/kg to 30 mg/kg (orhigher). These dosages may be administered daily, weekly, biweekly,monthly, or less frequently, for example, biannually, depending ondosage, method of administration, disorder or symptom(s) to be treated,and individual subject characteristics. Dosages can also be administeredvia continuous infusion (such as through a pump). The administered dosemay also depend on the route of administration. For example,subcutaneous administration may require a higher dosage than intravenousadministration.

In certain circumstances, it may be advantageous to formulatecompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited for the patient. Each dosage unitcontains a predetermined quantity of antibody calculated to produce atherapeutic effect in association with the carrier. The dosage unitdepends on the characteristics of the antibodies and the particulartherapeutic effect to be achieved.

Toxicity and therapeutic efficacy of the composition can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Antibodies that exhibit large therapeutic indices may be less toxicand/or more therapeutically effective.

The data obtained from the cell culture assays and animal studies can beused to formulate a dosage range in humans. The dosage of thesecompounds may lie within the range of circulating antibodyconcentrations in the blood, that includes an ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage composition form employed and the route of administration. Forany antibody used in the present invention, the therapeuticallyeffective dose can be estimated initially using cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofantibody which achieves a half-maximal inhibition of symptoms). Theeffects of any particular dosage can be monitored by a suitablebioassay. Examples of suitable bioassays include DNA replication assays,transcription-based assays, IL-22/IL-22R binding assays, IL-22/IL-10R2binding assays, IL-22/IL-22R/IL-10R2, and other immunological assays.

EXAMPLES Example 1 Selection of Anti-IL-22 scFv's

Selection of Parents GIL01 and GIL68

GIL01 and GIL68 were isolated from scFv libraries by soluble selectionon IL-22. Soluble selections were carried out using biotinylated IL-22with an N-terminal His/FLAG tagged protein (bio.IL-22H/F). Bio.IL-22H/Fwas initially used at a concentration of 100 nM. An scFv phagemidlibrary, which is an expanded version of the 1.38×10¹⁰ library described(Vaughan et al., 1996), was used to select antibodies specific forIL-22. Purified scFv phage (10¹² transducing units (tu)) were blockedfor 30 minutes in 100 μl 3% MPBS (3% milk powder in PBS), thenbio.IL-22H/F was added and incubated at room temperature for 1 hour.Phage/antigen was added to 50 μl of Dynal M280 Streptavidin magneticbeads, which had been blocked for 1 hour at 37° C. in 1 ml of 3% MPBS,then incubated for a further 15 minutes at room temperature. Beads werecaptured using a magnetic rack and washed 4× in 1 ml of 3% MPBS/0.1%(v/v) Tween 20 followed by three washes in PBS. After the last wash,beads were resuspended in 100 μl PBS and used to infect 5 mlexponentially growing E. coli TG-1 cells. Cells and phage on beads wereincubated for 1 hour at 37° C. (30 minutes stationary, 30 minutesshaking at 250 rpm), then spread on 2TYAG plates. Plates were incubatedat 30° C. overnight and colonies visualized the next day. Colonies werescraped off the plates into 10 ml 2TY broth and 15% glycerol added forstorage at −70° C.

Glycerol stock cultures from the first round panning selection weresuperinfected with helper phage and rescued to give scFvantibody-expressing phage particles for the second round of selection. Asecond and third round of soluble selection was carried out as describedabove, dropping the concentration of bio.IL-22H/F to 50 nM.

Isolation of Parents GIL16, GIL45, GIL60 and GIL92

GIL16, GIL45, GIL60 and GIL92 were isolated from scFv libraries by acombination of panning on an IL-22 fusion protein and soluble selectionon bio.IL-22H/F. Wells of a microtiter plate were coated with 10 μg/ml(Dulbecco's PBS, pH 7.4) human IL-22 fusion protein and incubatedovernight at 4° C. Wells were washed in PBS and blocked for 2 hours at37° C. in 3% MPBS. Purified phage (10¹² tu) in 100 μl of 3% MPBS wereadded to blocked wells and incubated at room temperature for 1 hour.Wells were washed 10 times with PBST (PBS containing 0.1% v/v Tween20),then 10 times with PBS. Bound phage particles were eluted with 100 μltrypsin solution (0.5 μg/ml trypsin in 50 mM Tris pH 8, 1 mM CaCl₂) for30 minutes at 37° C. The eluted phage were used to infect 10 mlexponentially growing E. coli TG1. Infected cells were grown in 2TYbroth for 1 hour at 37° C., as above, then streaked onto 2TYAG platesand incubated overnight at 30° C. Output colonies were scraped off theplates and phage rescued as described above. A second round of solubleselection was carried out as described above, using 100 nM bio.IL-22H/F.

Example 2 ScFv Blocks Binding of IL-22 to IL-22R

Inhibition assays were performed on the parent antibodies GIL01, GIL16,GIL45, GIL60, GIL68, and GIL92 to identify antibodies that block oralter binding of IL-22 to IL-22R and/or IL-22 receptor complex. CrudescFv containing periplasmic extracts were screened for the ability toinhibit the binding of bio.IL-22H/F to a human IL-22 receptor protein(hIL-22R). Output colonies from selections were picked into 96 wellplates containing 100 μl 2TYAG. ScFv production was induced by additionof 1 mM IPTG to exponentially growing cultures and overnight incubationat 30° C. Periplasmic extracts were prepared (Griffiths et al., 1993) in50 mM MOPS pH 7.4/0.5 mM EDTA/0.5M Sorbitol.

Microtiter plates were coated with 1.25 μg/ml of an IL-22 receptorprotein antibody (in PBS) for 1.5 hours at room temperature. Plates werethen washed three times in PBS, and blocked for 1 hour at roomtemperature with PBS containing 2% milk powder (2% MPBS). After afurther 3 washes, 50 μl of 25% cell conditioned medium containing IL-22receptor protein was added to each well, and incubated overnight at 4°C. The following day, 25 μl of sample and 25 μl of bio.IL-22H/F (54ng/ml in PBS/0.05% BSA/0.05% Tween) were added to the washed plates, andincubated for 1.5 hours at room temperature. After 3 washes in PBST,binding of bio.IL-22H/F was detected with Europium-Streptavidin and TRFdetected with the DELFIA® reagent kit and Victor 2™ Plate Reader (PerkinElmer).

Clones that showed inhibition of IL-22 binding were retested as purifiedscFv. Both the IL-22/IL-22R binding assay (described above) and theIL-22/IL-22 receptor complex assay (described below) were used. ScFvconcentrations were titrated in order to establish the clone potenciesas measured by assay IC₅₀ values. These were determined using GraphPadPrism software and four-parameter logistic equation curve fitting.Sample results from the IL-22 receptor complex assay are shown in FIG.1.

Example 3 Verification of IL-22 Binding by Phage ELISA

To establish the specificity of the scFv's for IL-22, a phage ELISA wasperformed using IL-22 fusion protein, IL-22H/F and an unrelated protein.Individual E. coli colonies containing phagemid were inoculated into 96well plates containing 100 μl 2TYAG medium per well. M13K07 helper phagewere added to a multiplicity of infection (moi) of 10 to theexponentially growing culture and the plates incubated an additional 1hour at 37° C. Plates were centrifuged in a benchtop centrifuge at 2000rpm for 10 minutes. The supernatant was removed and cell pellets wereresuspended in 100 μl 2TYAK and incubated at 30° C. overnight withshaking. The next day, plates were centrifuged at 2000 rpm for 10minutes and 100 μl phage-containing supernatant from each well wastransferred to a fresh 96 well plate. Phage samples were blocked in afinal concentration of 3% MPBS for 1 hour at room temperature.

Microtiter plates were coated with 1 μg/ml IL-22 fusion protein,IL-22H/F or an unrelated protein and incubated overnight at 4° C. Aftercoating, the solutions were removed from the wells, and the platesblocked for 1 hour at room temperature in 3% MPBS. Plates were rinsedwith PBS then 50 μl of pre-blocked phage was added to each well. Theplates were incubated at room temperature for 1 hour, then washed with 3changes of PBST followed by 3 changes of PBS. To each well, 50 μl of a1:5000 dilution of anti-M13-HRP conjugate (Pharmacia) was added and theplates incubated at room temperature for 1 hour. Plates were washed 3times with PBST then 3 times with PBS. Fifty μl of TMB substrate wasadded to each well and incubated until color development. The reactionwas stopped by the addition of 25 μl of 0.5 M H₂SO₄, and the absorbanceat 450 nm measured. These experiments confirmed the specific binding ofscFv clones to IL-22.

Example 4 Conversion of scF_(v) to IgG

Heavy and light chain V regions from scFv clones were amplified withclone-specific primers. PCR products were digested with appropriaterestriction enzymes and subcloned into vectors containing human IgG4heavy chain constant domain (for V_(H) domains) or vectors containinghuman lambda or kappa light chain constant domains as appropriate (V_(L)domains). The closest human germlines of the V_(H) and V_(L) segmentswere determined and this information was used to indicate whether kappaor lambda light chain constant domains were used (Table 4). Correctinsertion of V region domains into plasmids was verified by sequencingof plasmid. DNA from individual E. coli colonies. Plasmids were preparedfrom E. coli cultures by standard techniques and heavy and light chainconstructs co-transfected into HEK 293 EBNA cells using standardtechniques. Secreted IgG was purified using protein A sepharose(Pharmacia) and buffer exchanged into PBS.

TABLE 4 V_(H) and V_(L) germlines of IL-22 neutralizing clones CloneV_(H) germline V_(L) germline GIL01 3-11 (DP35) Vk1:L12 GIL16 1-18(DP14) Vk1:L12 GIL45 3-33 (DP50) VL2:2a2 (DPL11) GIL60 3-20 (DP32)VL2:2ac2 (DPL11) GIL68 1-2 (DP8) VL3:3h GIL92 1-2 (DP8) VL1:1e (DPL8)

Potency of purified IgG was verified in the biochemical IL-22 receptorcomplex inhibition assay as described below. IgG concentrations weretitrated in order to obtain potency values. Sample potency data is shownin Table 5.

TABLE 5 Potency of IL-22 scFv and IgG in the IL-22 receptor complexinhibition assay Clone ScFv potency (nM) IgG potency (nM) GIL01 104  13GIL16  49  10 GIL60  43  15 GIL68  7  2 GIL92  16 unobtainable GIL45 358180

Example 5 IL-22 Antibody Optimization

Large ribosome display libraries were created and screened for scFv'sthat specifically recognized recombinant human IL-22, essentially asdescribed in Hanes et al. (2000). Initially the five parent clones(GIL01, GIL16, GIL60, GIL68 and GIL92) were converted to ribosomedisplay format, and this template was subsequently used for librarycreation. On the DNA level, a T7 promoter was added at the 5′-end forefficient transcription to mRNA. On the mRNA level, the constructcontained a prokaryotic ribosome-binding site (Shine-Dalgarno sequence)and 5′ & 3′ stem loops for mRNA stability. At the 3′ end of the singlechain, the stop codon was removed and a portion of gill was added to actas a spacer, allowing folding of the scFv away from the ribosomal tunnel(Hanes et al. 2000).

Ribosome display libraries derived from the lead clones were created bymutagenesis of the single chain complementarity determining regions(CDRs) using PCR with non-proofreading Taq DNA polymerase. Affinitybased selections were performed where the library was incubated withbio.IL-22H/F, followed by streptavidin coated paramagnetic beads (DynalM280). Tertiary complexes (mRNA-ribosome-scFv) were recovered bymagnetic separation, while unbound complexes were washed away. The mRNAsencoding the bound scFvs were then rescued by RT-PCR as described (Haneset al., 2000) and the selection process repeated with decreasingconcentrations (100 nM-10 pM over 5 rounds) of bio.IL-22H/F.

Error prone PCR was introduced to further increase library size. Theerror rate that was employed created, on average, 7.2 mutations per1,000 bp after a standard PCR reaction based on the method of Cadwelland Joyce (1992). Initial error prone PCR reactions took place betweenthe first and second rounds of selection.

V_(H)/V_(L) recombination libraries for each parent clone were preparedfrom the V_(H) and V_(L) CDR ribosome display outputs after either thesecond or fourth round of selections. The V_(H) portion of the V_(H) CDRselection output for a particular lineage was specifically PCRamplified, using clone specific primers. The V_(L) portion of the V_(L)CDR selection output for the same lineage was amplified separately.These two PCR products were recombined via an overlapping PCR reaction.This created a complete library of scFv products containing allcomponents required for further rounds of ribosome display selection.

For some clones, phage display libraries were also utilized. Phagelibraries were created by mutagenesis of single chain CDRs using PCRreactions with appropriate primers, and selected as described above.These outputs were also combined with ribosome display selection outputsto create V_(H)V_(L) recombination libraries. The V_(H) selectionoutputs from the fourth round of ribosome display, together with theoutputs from the third round of phage display, were recombined with theV_(L) outputs from the same lineage. This was achieved using clonespecific primers and over-lapping PCR to produce V_(H)/V_(L)recombination libraries. Selections with soluble bio.IL-22H/F continuedin ribosome display format, as described above. The scFv regions ofselection outputs were directionally cloned into pCANTAB6 for productionof scFv for biochemical high throughput screening.

Example 6 Identification of Optimized Clones

Two assays were used for high throughput screening of selection outputs.Outputs derived from clones GIL01, GIL16 and GIL68 were screened in ahomogeneous time resolved fluorescence assay (HTRF®, CisBiointernational), while GIL60 and GIL92 outputs were screened in aDELFIA® (Perkin Elmer) assay.

HTRF® Epitope Competition Assay

Crude scFv containing culture supernatants from GIL01, GIL16 and GIL68output clones were prepared as described above and screened forinhibition of bio.IL-22H/F binding GIL68 in an HTRF assay.

Cryptate labeled GIL68 IgG (labeling kit from Cis Biointernational) wasdiluted 400 fold in assay buffer (PBS/0.4M KF/0.05% BSA/0.05% Tween),followed by the addition of 7.5 nM Streptavidin XL665 (Xlent, CisBiointernational). This solution was mixed with crude scFv sample(diluted 125×), and bio.IL-22H/F in a Packard black 384 well Optiplate(Perkin Elmer). Plates were incubated for 1 hour at room temperaturethen read using a Victor 2™ Plate Reader (Perkin Elmer). The 665 nM/620nM emission ratio was used to calculate the percentage of specificbinding in each well.

DELFIA® Time Resolved Fluorescence Assay

GIL60 and GIL92 output clones were screened for inhibition ofbio.IL-22H/F binding to an IL-22 receptor complex.

Microtiter plates were coated with IL-22 receptor complex antibody (1μg/ml in PBS), and incubated for 1.5 hours at room temperature. Plateswere washed three times in PBST, and blocked for 1 hour at roomtemperature with 2% MPBS. After a further 3 washes, diluted cellconditioned medium containing an IL-22 receptor complex was added andincubated overnight at 4° C. Crude scFv supernatants were prepared asdescribed above. The following day, 25 μl of diluted scFv sample and 25μl of bio.IL-22H/F (6 ng/ml) were added to the washed plates, andincubated for 1.5 hours at room temperature. Plates were washed 3 timesin PBST, then binding of bio.IL-22H/F to the IL-22 receptor complex wasdetected with Europium-Streptavidin and the DELFIA® reagent kit(PerkinElmer). Time Resolved Fluorescence was measured using a Victor 2™Plate Reader (Perkin Elmer).

Purified scFv from positive clones identified from the screening weretested in the DELFIA® IL-22 receptor complex competition assay asdescribed above. A titration of scFv concentrations was used in order toestablish the clone potency as measured by IC₅₀ values in the assay.Sample results are shown in FIG. 2. Fourteen optimized clones weredesignated 097D09, 062A09, 062G05, 087B03, 367D04, 368D04, 166B06,166G05, 375G06, 376B10, 354A08, 355B06, 355E04, and 356A11.

Example 7 Ranking of Optimized Clones in the BAF3-IL-22 ProliferationAssay

Proliferation assays were performed to assess the antibody's ability toblock the IL-22 mediated BaF3 cell proliferation. BaF3 cells expressinghIL22R/hIL10R2 were generated by co-transfection of BaF3 cells withhIL22R-GFP and hIL10R2-YFP. BaF3 cells expressing both hIL22R andhIL10R2 (BaF3-IL-22 receptor cells) were sorted and collected by FACS.

BaF3-IL-22 receptor cells were routinely maintained in RPMI1640 with 10%FBS and 1 ng/mL murine IL-3. Immediately before assay setup, cells werewashed 4 times in assay medium (RPMI1640 with 10% FBS, 100 U/mlPenicillin and 100 μg/ml Streptomycin), resuspended in assay medium andincubated at 37° C., 5% CO₂ for 6-8 hours. To prepare assay plates, 100μl of cells (1×10⁵/ml in assay medium) were added to the central 60wells of a 96 well flat-bottomed tissue culture plate (Costar). TestscFv or IgG samples were prepared by diluting the stock sample in assaymedium followed by filtration through a 0.22 μM filter. Serial 5-folddilutions of samples were prepared in a separate dilution plate. Cellcontaining wells were treated with 50 μl of sample followed by 50 μl ofhuman IL-22, (40 ng/ml in assay medium), and were then incubated for 40hours at 37° C. in 5% CO₂. Control wells included media alone and cellseither alone or in the presence of 10 ng/mL human IL-22.

Cell proliferation was detected by the addition of 20 μl of Alamar Blue(Serotec) to wells, followed by incubation for 5 hours±30 mins at 37° C.in 5% CO₂. Plates were mixed by gentle tapping to ensure even signalthroughout the wells before measurement of fluorescence (excitation=560nM, emission=590 nM). EC₅₀ and IC₅₀ values were estimated usingfour-parameter logistic curve fitting (Graphpad Prism 2 Software) andwere used to rank antibodies. Sample potency data for optimized scFvsand IgGs are shown in Table 6.

TABLE 6 IC₅₀ values of scFv and IgG clones in BaF3-IL-22 proliferationassay Clone Parent IC₅₀ of scFv (pM) IC₅₀ of IgG (pM) 097D09 GIL01 298 ±246 197 ± 42 062A09 GIL16 267  83 ± 37 062G05 GIL16 182 112 ± 30 087B03GIL60 212 105 ± 17 367D04 GIL60 160 ± 49 126 ± 6 368D04 GIL60 186 ± 66127 ± 10 166B06 GIL68 460  71 ± 23 166G05 GIL68 204  97 ± 23 375G06GIL68 118 ± 98 100 ± 1 376B10 GIL68 104 ± 47 119 ± 6 354A08 GIL92 219 ±83  79 ± 15* 355B06 GIL92 183 ± 3  92 ± 14* 355E04 GIL92 192 ± 47 100 ±14* 356A11 GIL92 124 ± 21  53 ± 5* *GIL92-derived clones were tested asgermlined IgGs.

Example 8 Germlining

Sequence data for the six parent clones was used to identify the nearestgermline sequence for the heavy and light chain of each clone.Appropriate mutations were made using standard site directed mutagenesistechniques with the appropriate mutagenic primers. Mutation of sequenceswas confirmed by sequence analysis. The sequences for the germlinedclones and their scFv and V_(H) and V_(L) domains are shown in Table 7.Purified scFv from the germlined parent clones were tested in thebiotinylated IL-22 binding IL-22 receptor complex competition assay asdescribed earlier, in order to establish the clone potency as measuredby IC₅₀ values in the assay. Results are summarized in Table 8.

TABLE 7A Amino Acid and Nucleotide Sequences of V_(H) and V_(L) Domains,F_(V), and CDRs of Germlined Antibodies (GIL01, GIL16, GIL45, GIL60,GIL68, GIL92, 062A09, and 087B03) GIL01 GIL16 GIL45 GIL60 GIL68 GIL92062A09 087B03 Region Type SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQID SEQ ID V_(H) AA NO: 365 NO: 383 NO: 401 NO: 419 NO: 437 NO: 455 NO:473 NO: 491 V_(L) AA NO: 366 NO: 384 NO: 402 NO: 420 NO: 438 NO: 456 NO:474 NO: 492 scF_(v) AA NO: 367 NO: 385 NO: 403 NO: 421 NO: 439 NO: 457NO: 475 NO: 493 H1 AA NO: 368 NO: 386 NO: 404 NO: 422 NO: 440 NO: 458NO: 476 NO: 494 H2 AA NO: 369 NO: 387 NO: 405 NO: 423 NO: 441 NO: 459NO: 477 NO: 495 H3 AA NO: 370 NO: 388 NO: 406 NO: 424 NO: 442 NO: 460NO: 478 NO: 496 L1 AA NO: 371 NO: 389 NO: 407 NO: 425 NO: 443 NO: 461NO: 479 NO: 497 L2 AA NO: 372 NO: 390 NO: 408 NO: 426 NO: 444 NO: 462NO: 480 NO: 498 L3 AA NO: 373 NO: 391 NO: 409 NO: 427 NO: 445 NO: 463NO: 481 NO: 499 V_(H) DNA NO: 374 NO: 392 NO: 410 NO: 428 NO: 446 NO:464 NO: 482 NO: 500 V_(L) DNA NO: 375 NO: 393 NO: 411 NO: 429 NO: 447NO: 465 NO: 483 NO: 501 scF_(v) DNA NO: 376 NO: 394 NO: 412 NO: 430 NO:448 NO: 466 NO: 484 NO: 502 H1 DNA NO: 377 NO: 395 NO: 413 NO: 431 NO:449 NO: 467 NO: 485 NO: 503 H2 DNA NO: 378 NO: 396 NO: 414 NO: 432 NO:450 NO: 468 NO: 486 NO: 504 H3 DNA NO: 379 NO: 397 NO: 415 NO: 433 NO:451 NO: 469 NO: 487 NO: 505 L1 DNA NO: 380 NO: 398 NO: 416 NO: 434 NO:452 NO: 470 NO: 488 NO: 506 L2 DNA NO: 381 NO: 399 NO: 417 NO: 435 NO:453 NO: 471 NO: 489 NO: 507 L3 DNA NO: 382 NO: 400 NO: 418 NO: 436 NO:454 NO: 472 NO: 490 NO: 508

TABLE 7B Amino Acid and Nucleotide Sequences of V_(H) and V_(L) Domains,F_(V), and CDRs of Germlined Antibodies (166B06, 166G05, 354A08, 355B06,355E04, 356A11, and 368D04) 166B06 166G05 354A08 355B06 355E04 356A11368D04 Region Type SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDV_(H) AA NO: 509 NO: 527 NO: 545 NO: 563 NO: 581 NO: 599 NO: 617 V_(L)AA NO: 510 NO: 528 NO: 546 NO: 564 NO: 582 NO: 600 NO: 618 scF_(v) AANO: 511 NO: 529 NO: 547 NO: 565 NO: 583 NO: 601 NO: 619 H1 AA NO: 512NO: 530 NO: 548 NO: 566 NO: 584 NO: 602 NO: 620 H2 AA NO: 513 NO: 531NO: 549 NO: 567 NO: 585 NO: 603 NO: 621 H3 AA NO: 514 NO: 532 NO: 550NO: 568 NO: 586 NO: 604 NO: 622 L1 AA NO: 515 NO: 533 NO: 551 NO: 569NO: 587 NO: 605 NO: 623 L2 AA NO: 516 NO: 534 NO: 552 NO: 570 NO: 588NO: 606 NO: 624 L3 AA NO: 517 NO: 535 NO: 553 NO: 571 NO: 589 NO: 607NO: 625 V_(H) DNA NO: 518 NO: 536 NO: 554 NO: 572 NO: 590 NO: 608 NO:626 V_(L) DNA NO: 519 NO: 537 NO: 555 NO: 573 NO: 591 NO: 609 NO: 627scF_(v) DNA NO: 520 NO: 538 NO: 556 NO: 574 NO: 592 NO: 610 NO: 628 H1DNA NO: 521 NO: 539 NO: 557 NO: 575 NO: 593 NO: 611 NO: 629 H2 DNA NO:522 NO: 540 NO: 558 NO: 576 NO: 594 NO: 612 NO: 630 H3 DNA NO: 523 NO:541 NO: 559 NO: 577 NO: 595 NO: 613 NO: 631 L1 DNA NO: 524 NO: 542 NO:560 NO: 578 NO: 596 NO: 614 NO: 632 L2 DNA NO: 525 NO: 543 NO: 561 NO:579 NO: 597 NO: 615 NO: 633 L3 DNA NO: 526 NO: 544 NO: 562 NO: 580 NO:598 NO: 616 NO: 634

TABLE 8 ScFv potencies of ungermlined and germlined parent clones in theIL-22 receptor competition assay Average IC₅₀ nM in IL-22 competitionParent clone assay scFv Parent Fully germlined GIL01 124 ± 50 143 ± 45GIL16 44 ± 1 38 ± 1 GIL60  51 ± 16 82 ± 3 GIL68  9 ± 1 14 ± 1 GIL92 18 ±2  40 ± 11

Nine of the optimized antibodies were germlined as described above.Eight germlined IgGs were tested in the BaF3-IL-22 proliferation assayas described above. Antibody IC₅₀ values from a representativeexperiment are shown in Table 9.

Antibody sequences were then sent to GENEART North America (28 KirkBradden Rd. East, Toronto, ON, Canada M8Y2E6), where they weresynthesized for optimized expression in CHO cells using GENEART'sproprietary optimization algorithm.

TABLE 9 lgG potencies of germilned optimized clones in the BaF3-IL-22Rproliferation assay IC₅₀ (pM) of non- IC₅₀ (pM) of Clone Parentgermlined lgG germlined lgG 087B03 GIL60 72 ± 6  118 ± 19 166B06 GIL68109 ± 16  169 ± 32 166G05 GIL68 366 ± 226* 109 ± 31 356A11 GIL92 ND 53 ±5 355B06 GIL92 ND  92 ± 14 355E04 GIL92 ND 100 ± 14 354A08 GIL92 ND  79± 15 062A09 GIL16 108 ± 23  unobtainable *sample contained precipitateND = not determined

Example 9 Antibody Inhibits IL-22 Induced GROa Secretion from HT29 Cells

GROa assays were performed to assess the antibody's ability to block theIL-22 induced GROa secretion from HT29 cells. HT29 cells were seeded in96 well flat bottom tissue culture plate (Corning Inc. Cat. #3595) inDMEM medium (DMEM+10% FBS+100 unit/ml penicillin and streptomycin+2 mMGlutamine) at 5×10⁴/well. 10 ng/ml IL-22 was mixed with serially dilutedantibody in DMEM medium and incubated for 30 min at 37° C. 24 hoursafter seeding, medium was removed from HT29 cells and pre-mixed IL-22and antibody were added to the cells in 96 well plate.

After 48 hours of incubation at 37° C. with 5% CO₂, medium was collectedand secreted GROa was tested using Human GROa Immunoassay kit (R&DSystems, Cat. DGR00), according to the manufacturer's directions.Results are presented in FIG. 3.

Example 10 Antibody Binds to and Inhibits Different Species IL-22

Cross species reactivity of germlined and non-germlined optimizedantibodies were determined as follows: ELISA plates (Costar, Cat. #3590)were coated overnight with 1 μg/ml of rat, mouse, or human IL-22 orhuman IL-26 in PBS buffer. Plates were washed with PBST buffer (0.05%Tween20 in PBS) 3 times, then blocked with 1% BSA (Sigma A8918)/PBST for1 hr at RT. Antibodies were added at 1 μg/ml, incubated 1 hr at 25° C.The plates were washed, then HRP-conjugated goat anti-human IgG antibody(Southern Biotech Association, Cat. #2040-05) was added. The plates wereincubated for 1 hour at 25° C., then washed with PBST, and developedwith TMB (KPL, Cat. #50-76-04). Reaction was stopped with 0.18 M H₂SO₄Plates were read at OD 450 nm. Results are presented in FIG. 4.

These antibodies were also evaluated in both the GROa cell assay andBaF3-IL-22 proliferation assay. As shown in Tables 10(a) and 10(b), theantibodies blocked the activity of human, monkey, rat, and mouse IL-22signalling via a human IL-22 receptor. 356A11 and 368D04 alsodemonstrated cross-species reactivity against murine, rat, and monkeyIL-22 using real-time biospecific interaction analysis (BIA), asdiscussed further in Example 11.

TABLE 10(a) IL-22 antibodies are highly potent for blocking otherspecies of IL-22 as shown in the GROa cell based assay system. Valuesshown represent IC₅₀ values in pM. Protein ID human IL-22 murine IL-22rat IL-22 monkey IL-22 356A11 123.64 143.76 210.91 89.57 368D04 154.07156.25 281.12 184.10 control 1 353.18 468.34 1161.57 343.19 control 21955.80 3399.79 10697.17 1459.27

TABLE 10(b) IL-22 antibodies are highly potent for blocking otherspecies of IL-22 as shown in the BaF3 cell based assay system. Valuesshown represent IC₅₀ values in pM. Protein ID human IL-22 murine IL-22rat IL-22 monkey IL-22 356A11 3.57 2.53 10.69 2.58 368D04 3.63 1.4712.07 3.87 control 1 6.40 5~6 27.37 7.18 control 2 204.98 1033.262500.00 134.27

Example 11 Comparison of Binding Kinetics Between Rat Anti-IL-22Monoclonal Antibodies and Human Anti-IL-22 Monoclonal Antibodies

The binding kinetics of human, monoclonal anti-IL-22 antibodies (356A11and 368D04) and rat, monoclonal anti-IL-22 antibodies (P3/3 (Ab-02) andP3/2 (Ab-04) from WO 2005/000897 and WO 02/068476) to human IL-22 wereevaluated by real-time biospecific interaction analysis (BIA) usingsurface plasmon resonance technology.

To prepare the biosensor surface for the rat monoclonal antibodies,Protein A/G (Pierce #21186, Rockford, Ill.) was immobilized onto aresearch-grade carboxymethyl dextran chip (CM5) using amine coupling.The surface was activated with EDC/NHS. The protein A/G was injected ata concentration of 50 μg/ml in sodium acetate buffer (pH 4.0). Theimmobilization was done using the wizard tools with aim of 3000 (RUs)for the protein A/G. Remaining activated groups were blocked with 1.0 Methanolamine (pH 8.0). The first flow cell was used as a referencesurface to correct for bulk refractive index, matrix effects, andnon-specific binding. The second, third, and fourth flow cells werecoated with the capturing molecule. The rat monoclonal antibodies Ab-02and Ab-04, which bind to protein A/G, were captured onto the protein A/Gsurface by injecting 30 μl of a 1 μg/ml solution. The net differencebetween the baseline and the point approximately 90 seconds aftercompleting Ab-02 or Ab-04 injection was used to represent the amount ofligand bound.

To prepare the biosensor surface for the human monoclonal antibodies,either human monoclonal antibody (356A11 or 368D04) or control antibodywere immobilized onto a research-grade carboxymethyl dextran chip (CM5)using standard amine coupling. The surface was activated with EDC/NHS.The capturing antibodies were injected at a concentration of 1 μg/ml insodium acetate buffer (pH 5.5). Remaining activated groups were blockedwith 1.0 M ethanolamine (pH 8.0). The first flow cell was used as areference surface to correct for bulk refractive index, matrix effects,and non-specific binding. The second, third, and fourth flow cells werecoated with the capturing molecule.

For Ab-02 and Ab-04, solutions of human IL-22 at 300, 100, 50, 25, 12.5,6.4, 3.2, 1.6 and 0 nM concentrations were injected in triplicates at aflow rate of 30 μl per minute for 3 minutes and the amount of boundmaterial as a function of time was recorded as sensorgrams. Thedissociation phase was monitored in HBS/EP buffer for 10 minutes at thesame flow rate followed by a 5 μl injection of 0.1% TFA and a 5 μlinjection of glycine pH 1.5 to regenerate a fully active capturingsurface.

For 356A11 and 368D04, solutions of human IL-22 at 400, 200, 100, 50,25, 12.5, 6.25 and 0 nM were injected in triplicates at a flow rate of100 μl per minute (high flow to avoid non specific binding) for 3minutes, and the amount of bound material as a function of time wasrecorded as sensorgrams. The dissociation phase was monitored in HBS/EPbuffer for 60 minutes at the same flow rate followed by two 5 μlinjections of glycine pH 1.5 to regenerate a fully active capturingsurface.

All kinetic experiments were done at 22.5° C. in HBS/EP buffer. Blankand buffer effects were subtracted for each sensorgram using doublereferencing. In control experiments the first injection containedbuffer.

The kinetic data were analyzed using BIAevaluation software 3.0.2applied to a 1:1 model. The apparent dissociation (K_(d)) andassociation (K_(a)) rate constants were calculated from the appropriateregions of the sensorgrams using a global analysis. The affinityconstants of the interaction between antibody and analyte werecalculated from the kinetic rate constants by the following formulae:K_(D)=K_(d)/K_(a), where K_(D) is the dissociation constant, andK_(A)=K_(a)/K_(d), where K_(A) is the association constant. The bindingdata for Ab-02 and AB-04 are summarized in Tables 11A and 11B. Thebinding data for 356A11 and 368D04 are summarized in Table 12.

TABLE 11A Kinetic parameters for the interaction between human IL-22 andanti-IL-22 antibodies Ab-02 and Ab-04 Ab-02 Ab-02 Ab-04 Ab-04 k_(a)(M⁻¹s⁻¹) k_(d) (s⁻¹) k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) Protein A/G 2.78 E+051.45 E−03 5.15 E+05 1.23 E−03

TABLE 11B Kinetic data of rat monoclonal antibodies for human IL-22Antibody Ka (1/Ms) Kd (1/s) KA (1/M) KD (M) Chi2 Ab-02 2.78 E+05 1.45E−03 1.92 E+08 5.22 E−08 0.49 Ab-04 5.15 E+05 1.23 E−03 4.22 E+08 2.38E−09 0.53

TABLE 12 Kinetic data of human monoclonal antibodies for human IL-22Antibody Ka (1/Ms) Kd (1/s) KA (1/M) KD (M) Chi2 356A11 7.91 E+04 4.27E−06 1.85 E+10 5.40 E−11 0.223 368D04 1.89 E+05 2.50 E−05 7.56 E+09 1.32E−10 0.298

These results show that the human monoclonal anti-IL-22 antibodies ofthis invention have a significantly higher affinity for human IL-22 thanthe rat monoclonal anti-IL-22 antibodies Ab-02 and Ab-04, described inWO 2005/000897 and WO 02/068476 as having the ability to neutralizehuman IL-22. Specifically, the dissociation constant of 356A11(K_(D)=5.40×10⁻¹¹ M or 0.054 nM) for human IL-22 is approximately1000-fold and more than 40-fold greater than the dissociation constantsof Ab-02 (K_(D)=5.22×10⁻⁸ M or 52 nM) and Ab-04 (K_(D)=2.38×10⁻⁹ M or2.38 nM), respectively. Similarly, 368D04 (K_(D)=1.32×10⁻¹¹ M or 0.132nM) has an approximately 400-fold and 18-fold stronger affinity forhuman IL-22 than Ab-02 and Ab-04, respectively. The binding profiles of356A11 and 36804 for monkey, murine, and rat IL-22 were similar to thatof human IL-22 (data not shown).

The binding specificities of 356A11 and 368D04 were also evaluated usingBIA. Neither antibody showed cross reactivity with human IL-10, humanIL-19, human IL-20, human IL-24, human IL-28A, human IL-29, humanIFN-α2c, or human IFN-ω (data not shown).

Example 12 In Vivo Half Life of Anti-Human IL-22 Antibodies

The anti human IL-22 antibodies of the invention have long in vivo halflives. For example, the in vivo half life of both 356A11 and 368D04 inDBA/1 mice was eight days. Specifically, a single dose of either 356A11or 368D04 (16 mg/kg) was administered intraperitoneally to DBA/1 mice.The 356A11 and 368D04 antibodies were detected in serum from the miceusing a human IgG1 ELISA. The time courses of 356A11 and 368D04 serumconcentrations are shown in FIGS. 11A and B. The PK parameters of 356A11and 368D04 are summarized in the Table 13 below.

TABLE 13 PK Parameters of 3536A11 and 368D04 PK Parameter 356A11 368D04AUC (ng.hr/mL) 57273471 54052982 Cmax (ng/mL) 169854 177161 Tmax (hr) 4824 t1/2 (hr) 192 206 t1/2 (days) 8 8.6

Example 13 Treatment of Arthritis

Arthritis is a disease characterized by inflammation in the joints.Rheumatoid Arthritis (RA) is the most frequent form of arthritis,involving inflammation of connective tissue and the synovial membrane, amembrane that lines the joint. The inflamed synovial membrane ofteninfiltrates the joint and damages joint cartilage and bone. Both IL-22and IL-22R protein and/or transcript are associated with human disease.In RA synovial biopsies, IL-22 protein is detected in vimentin⁺ synovialfibroblasts and some CD68⁺ macrophages while IL-22R is detected insynovial fibroblasts. Treatment of synovial fibroblasts with IL-22induces the production of monocyte chemoattractant protein-1, MCP-1, aswell as general metabolic activity (Ikeuchi, H. et al. (2005) ArthritisRheum. 52:1037-46).

IL-22 is used to study its effect on cells from the synovial membrane,the membrane that lines the joints. Human fibroblast-like synoviocytes(HFLS) (Cell Applications (San Diego, Calif.)) are isolated fromsynovial tissues of rheumatoid arthritis patients undergoing jointsurgery. HFLS are cultured with human IL-22 for 48 hours, and thesupernatants are removed and tested for chemokines and cytokines byELISA. IL-22 will increase HFLS secretion of chemokines MCP-1, Eotaxin,and IP-10, and cytokines TNFα, IL-6, and IL-8. These chemokines andcytokines are known in the art to promote inflammation through a numberof activities, and increased concentrations in the joints caused byIL-22 exacerbates inflammation and RA.

The ability of human anti IL-22 antibody to ameliorate symptoms incollagen induced arthritis (CIA) was examined using the 356A11 and368D04 antibodies. CIA is the standard mouse and rat model for studyingrheumatoid arthritis, see e.g., Holmdahl et al., (2002) Ageing Res.Rev., 1:135. On day 0, male DBA/1 (Jackson Laboratory, Bar Harbor, Me.)mice were injected subcutaneously in the base of the tail with 100 μg ofbovine Collagen Type II (Chondrex, Redmond, Wash.) in complete Freund'sadjuvant, and on day 21, the mice were boosted with 100 μg of bovineCollagen Type II in incomplete Freund's adjuvant.

Mice were monitored at least two times a week for disease progression.The disease severity was scored using gross paw evaluation as follows:0=no swelling, 1=1 to 2 swollen digits or swollen ankle, 2=more than 2swollen digits or mild paw swelling, 3=extensive paw swelling, and4=ankylosis of paw. Mice injected with an isotype control antibody afterthe collagen injections progressively developed disease. Treatment witheither 356A11 or 368D04 significantly reduced disease progression. Thesewere blind studies so the investigators did not know which animal groupreceived which antibody until the end of the study.

Various doses of 356A11 and 368D04 were evaluated. The treatments of356A11 or 368D04 (or a human IgG1λ isotype control antibody) wereinitiated when 10% of the mice in the treatment group had a diseaseseverity score of at least 1. The antibody was administered at variousfrequencies: every other day, once a week, or twice a week, and the micewere monitored for disease progression. Administering either 8 or 16 mgkg⁻¹ of 356A11 every other day significantly blocked progression of CIAin multiple studies as shown in FIGS. 12A-B, 13, and 40-41. Fourseparate CIA studies (FIGS. 13A-D), testing with 8 mg kg-1 of 356A11every other day, show that the 356A11 antibody consistently blockeddisease progression in the murine CIA model. Surprisingly, twice weeklyand even weekly dosing with 8 mg kg⁻¹ of 356A11 was also sufficient inmultiple studies to significantly block disease progression as shown inFIGS. 14-15, 41, and 44A-B.

In the first CIA study with 368D04 (administered every other day at 16mg kg⁻¹), little or no efficacy was observed. However, administering 8mg kg⁻¹ of 368D04 once a week in multiple studies significantly blockeddisease progression as shown in FIGS. 46A-C.

The human anti IL-22 antibody's ability to significantly block diseaseprogression when administered once a week in the CIA model indicatesthat the antibody could be administered with a similar dosing frequency,or an even further extended dosing frequency, such as once every twoweeks, when administered in humans.

Treatment effects of the anti-IL-22 antibody were also evaluated byhistopathological analysis of the paws. At the end of the study, animalswere euthanized, paws were harvested and fixed with 10% formalin forhistology, decalcified, and embedded in paraffin for sectioning andstandard H&E staining. The paws were scored on a 5-grade scoring method(0-4) to characterize intensity and extent of arthritis. Inflammatoryinfilitrates were used for scoring in addition to other changes relatedto the inflammation, such as pannus formation, fibrous of the synovialmembrane, articular cartilage erosin and/or subchondral bonedestruction. Histology grades were determined using readings ofindividual paws: NAD=0 or nothing abnormal discovered; 1=slight tomoderate; 2=miled to moderate; 3=marked; 4=massive. The histologicaleffect of the therapeutic administration of the anti-IL-22 antibodiesare shown in FIGS. 16A-F, 42A-C, 43, and 45A-B. As shown in FIGS. 16A-F,42A-C, 43, and 45A-B, administering 356A111 in multiple studiesameliorated symptoms of collagen induced arthritis in mice. Similarly,administering 368D04 every other day at 8 mg kg⁻¹ in multiple studiesameliorated symptoms of collagen induced arthritis in mice. FIGS. 47A-C.

In addition to histopathological evaluations, bone destruction intreated mice was also examined. At the end of the study, paws were fixedin a lateral position and X-ray pictures were taken with a Faxitronmachine. The Faxitron provides high resolution x-ray radiographs, andthe high magnification capability provides enhanced imaging performance.The Faxitron radiographs correlated with visual gross paw evaluationscores and showed that treatment with anti-IL-22 antibody prevented bonedestruction as compared to treatment with the isotype control antibody(data not shown).

Example 14 Detection of Serum IL-22 by ELISA and Increased Detection ofSerum IL-22 In Vivo with 356A11 Treatment

To date, the detection of IL-22 gene expression has only been reportedat the RNA level. With the ELISA described below, IL-22 is not detectedin normal mice. We have discovered, though, that this ELISA allows thedetection of IL-22 in the circulation of arthritic mice. Furthermore,administration of the 356A11 antibody enables the detection of IL-22 atten-fold higher levels. The 356A11 antibody at the doses givensequesters the cytokine, thus neutralizing its activity as shown inprior examples. With the circulation of this antibody within the bloodstream, the antibody-sequestered IL-22 is now detected at higher levelswith this ELISA. This is due to the unique and distinct epitopes of thecapture and detector antibodies that constitute this mIL-22 ELISA. ThisELISA detects IL-22/356A11, IL-22BP/IL-22, IL-22BP/356A11/IL-22 andnaked IL-22, equivalently.

In the context of the CIA studies discussed previously (Example 13),arthritic mice were treated every other day with 16 mg kg⁻¹ of 356A11antibody. At day 30 after the collagen boost, serum was collected fromthe mice after sacrifice. Levels of IL-22 in the serum samples were thenmeasured by ELISA.

For this ELISA format, 1 mGIL19P3/1, a rat IgG1k monoclonal anti-murineIL-22 antibody, was coated on a 96-well microtiter ELISA plate to beused as the capture antibody. Serum samples obtained from arthritic micewere serially diluted and then added to the coated microtiter plates.After allowing the samples to incubate with 1 mGIL19P3/1 in themicrotiter plates, the plates were washed and 2hmGIL19P3/5-bio, a ratIgG2ak monoclonal anti-IL-22 antibody conjugated to biotin was added tothe plates as a detection antibody. After another incubating and washingstep, streptavidin conjugated to horseradish peroxidase (HRP), whichconverts tetramethylbenzidine (TMB) to a blue pigment and isquantifiable with a spectrophotometer, was added, incubated and theplate washed. After the final incubation with TMB followed by additionof diluted H₂SO₄ to stop the enzymatic reaction, absorbance was measuredat 450 nm using a spectrometer. Alternatively, and because the detectorantibody's isotype, rat IgG2ak, is distinct from the coat antibodyisotype, an HRP-conjugated secondary antibody that binds to rat IgG2aantibody can also be used. Using this sandwich ELISA, we demonstratedthat the addition of increasing amounts of either mIL-22BP and/or 356A11does not affect the ability to detect a fixed concentration of murineIL-22. This ELISA has been used to detect IL-22 in the serum of miceafter an i.p., LPS injection.

When CIA mice were administered the 356A11 antibody, as indicated above,IL-22 was detected in the serum at levels ranging from about 1 ng/mL toabout 9 ng/mL. See FIG. 17. Conversely, the arthritic mice in thecontrol treatment group, receiving a human IgG1λ isotype controlantibody, recorded significantly lower in vivo levels of IL-22 (lessthan 1 ng/mL). See FIGS. 17 and 18. Thus, the 356A11 antibody capturesIL-22 in vivo to produce a stabilized antibody/cytokine complex thatcirculates. This in turn permits the detection of in vivo IL-22 withincreased sensitivity as shown in FIG. 16. In contrast to the largeamounts of antibody used in the above study, it is proposed that 356A11at considerably lower levels could also be administered and have thesame effect.

Example 15 Treatment of Psoriasis

Levels of IL-22 and IL-22R RNA were measured in paired tissue samples(lesion vs. non-lesion) from human psoriatic patients using quantitativePCR. This study demonstrated that levels of IL-22 and IL-22R wereupregulated in psoriatic lesions. FIG. 19. Other evidence implicatesIL-22 in the development of psoriasis. For example, transgenic mice thatconstitutively express IL-22 present with thick skin, mononuclear immunecell infiltrates, characteristic of psoriatic lesions, and die soonafter birth. WO 03/083062. Similarly, administering IL-22 to miceinduces thickening of skin and mononuclear immune cell infiltrates. WO03/083062. IL-22 also induces human keratinocyte hyerplasia, suggestingan important role in skin inflammatory processes. Boniface et al., J.Immunol., 2005 174:3695-3702.

Xenogenic transplantation in SCID mice is a recognized model forstudying psoriasis, see e.g., Boehncke et al. Br. J. Dermatol. 2005,153(4):758-66. Under local anesthesia, lesional split-skin (thicknessabout 0.5 mm) is exised from a patient with chronic plaque-stagepsoriasis. Human split grafts are transplanted on the back of 6-8 weekold SCID mice. Mice are given 3 weeks to accept the graft and heal. At22 days following transplantation, mice are injected intraperitoneallywith 8 mg kg⁻¹ of human anti-11-22 antibodies, such as germlined 087B03,368D04, 354A08 or 356A11, every other day. As a negative control, micereceive daily intragastric applications of 200 μL PBS. As a positivecontrol, mice receive daily intragastric application of 2 mg kg⁻¹dexamethasone in 200 μL PBS. The negative controls develop hallmarks ofpsoriasis, including acanthosis, papillomatosis, parakeratosis, and adense mononuclear infiltrate. Mice are sacrificed at day 50 followingtransplantation and the grafts with surrounding skin are excised. Onehalf of the graft is fixed in formalin and the other half is frozen inliquid nitrogen. Routine hematoxylin and eosin stainings are performedand the pathological changes of the grafts are analysed bothqualitatively (epidermal differentiation) and quantitatively (epidermalthickness, inflammatory infiltrate). The mean epidermal thickness may bemeasured from the tip of the rete ridges to the border of the viableepidermis using an ocular micrometer. The density of the inflammatoryinfiltrate may be determined by counting the number of cells in threeadjacent power fields. Disease progression may be evaluated usinghistological analysis to measure hallmarks of psoriasis, such asacanthosis, papillomatosis, parakeratosis, inflammatory infiltrates, andthe appearance of the corneal and granular layers.

Negative control mice injected with 200 μL PBS or an isotype-matchedcontrol antibody following graft transplantation progressively developpsoriasis. Because psoriatic lesions express higher levels of IL-22,treatment with anti-IL-22 antibodies, for example with germlined 087B03,368D04, 354A08 or 356A11, is expected to suppress or delay psoriasis, asconfirmed in the adoptive transfer study reported below.

Adoptive transfer of CD4+CD45rb^(high) Tcells in scid/scid mice isanother recognized model for studying psoriasis in mice. Hong et al., J.Immunol., 162(1):7480-91 (1999). In this model, coadministration of LPSand IL-12 or staphylococcal enterotoxin B into scid/scid mice 1 dayafter adoptive transfer of CD4+CD45rb^(high) Tcells induces skin lesionsexhibiting the hallmarks observed in human psoriasis.

Using a slight modification of this model, CD4+ CD45rb^(high)CD25−TCELLS were transferred to scid/scid mice with or withoutcoadministration of LPS and IL-12 at day one following adoptivetransfer. CD4+CD45rb^(high)CD25− Tcells were able to induce psoriaticlesions when transferred into scid/scid mice even when they wereadministered without LPS and IL-12. Thus, administration of IL-12 andLPS following adoptive transfer did not seem to alter the efficacy ofanti IL-22 antibodies in this model.

Cells for adoptive transfer were prepared as described before withslight modification. Spleens were collected from 6- to 8-wk-old BALB/cBydonor mice (Jackson Laboratory, Bar Harbor, Me.) and splenocytes wereisolated by mechanical homogenization of whole spleens. CD4⁺ T cellswere selected using murine CD4 enrichment kit (R&D) according to themanufacturer's instruction. CD4 enriched T cells were labeled withanti-CD4-PE (Pharmingen), anti-CD45RB-FITC and anti-CD25-APC(Pharmingen). Cells were sorted using a Moflo (Becton Dickinson, SanJose, Calif.) cell sorter. CD4+CD45Rb^(high)CD25− cells were collectedand were >95% pure. Cells were re-suspended in saline at 2×10⁶ cells/mland 4×10⁵ cells were injected i.p. into C.B-17/Prkdc scid/scid mice(Jackson Laboratory, Bar Harbor, Me.). In some cases, 10 ng IL-12 and 20μg LPS were administrated i.p. to the recipient mice that receivedCD4⁺CD45Rb^(high) cells on day 1 and an additional dose of IL-12 wasadministered on day 3. Mice were dosed with 16 mg/kg of either anisotype control antibody or anti-IL-22 antibodies (356A11 or 368D04) onthe day of adoptive transfer and once a week thereafter for 10 weeks.Mice were monitored for clinical symptoms of skin lesions twice a week.At termination of the study, mouse ear, skin, lymph nodes, and spleenwere harvested for further ex-vivo studies.

Clinical Evaluation

Mice were evaluated by blinded investigators twice per week starting 10days post adoptive transfer. To record disease progressionsemiquantitative clinical scores from 0 to 6 were given based onphysical appearance: 0=no skin or ear symptoms; 0.5=slight erythema onear or eye lid, 1=mild, moderate erythema on ears or eyelids with mildthickening of the ear (<2% of the body surface); 2=moderate to severeerythema on 2-10% of the body surface, mild scaling; 3=severe erythemaand scaling on 10%-20% of the body surface; 4=very severe, extensiveerythema and scaling on 20%-40% of the body surface. 5=very severe,extensive erythema and scaling on 40%-60% of the body surface. 6=verysevere, extensive erythema and scaling and scaling on greater than 60%of the body surface. Specific observations were noted based on furcondition, ear manifestations, eyelid appearance, and presence ofinflammation on limbs and tail.

In the study without coadministration of LPS and IL-12, antibodies356A11 and 368D04 significantly suppressed skin inflammation, whencompared to control antibody treated mice, starting as early as day 21for 356A11 and day 35 for 368D04 (FIGS. 20A-B). Similar results wereobserved in the study with coadministration of LPS and IL-12 at day onefollowing adoptive transfer, with 356A11 and 368D04 significantlysuppressing skin lesions, when compared to the control antibody,starting as early as day 28 post adoptive transfer. (FIGS. 27A and B).In a separate study, 356A11, without (FIGS. 34A-B) or with (Figures and37A-B) coadministration of LPS and IL-12 at day one following adoptivetransfer, significantly suppressed skin inflammation, when compared tocontrol antibody treated mice, starting as early as day 21.

Cytokine Detection

Mouse serum or supernatants from the cell culture were quantitated forIL-6, IFNγ, and TNFα using an ELISA kit (R&D system). ELISA for IL-22,IL-17A, IL-17F, and IL-17A/F heterodimer involved coating a 96-wellflat-bottom plate (Costar) overnight at 4° C. with 100 μl of a 2 μg/mlsolution of anti-1 L-22 (Ab-01), anti-IL-17A (R&D), or anti-IL-17F(RK015-01) antibody in PBS. Plates were then washed with PBS/Tween(0.05% Tween-20 in PBS) and blocked with 200 μl PBS plus 1% BSA for 1 hat RT. Antibodies to murine IL-22 (Ab-01, Ab-02, and Ab-03) and murineIL-17F (RK015-01) were generated by methods described in Li et al., Int.Immunopharmacol. 4:693-708 (2004). In between all of the followingsteps, plates were washed with PBS/Tween. IL-22 (R&D), IL-17A (R&D),IL-17F, and IL-17A/F standards, sample supernatants and serum samples(diluted with PBS 1:5) were then added. Biotinylated secondaryantibodies for anti-IL-22 (Ab-03), anti-IL-17A (R&D), and anti-IL-17F(RK015-01) were then added to the respective plates at 0.5 μg/ml in 1%BSA/PBS solution and incubated for 1 h at 37° C. Poly HRP-labeledstreptavidin (Pierce) was added according to manufacturer's instructionfor 15 minutes. The plate was developed by adding TMB substrate for15-30 minutes. Assay was then read on a Molecular Devices (Sunnyvale,Calif.) plate reader and data were analyzed using SOFTmax software.

In the study without coadministration of LPS and IL-12, higher levels ofserum IL-22 were detected in mice treated with 356A11 than in micetreated with control antibody, indicating that 356A11 captures andstabilizes IL-22 in vivo (FIG. 21), as discussed in Example 14. Similarresults were also observed in 356A11 treated mice with coadministrationof LPS and IL-12 at day one following adoptive transfer. (FIG. 28).These results were replicated in a separate study with 356A11 without(FIG. 35) and with (FIG. 38) coadministration of LPS and IL-12 at dayone following adoptive transfer. Serum IL-22 was not detected in 368D04treated mice (FIG. 21).

In the study without coadministration of LPS and IL-12, significantlylower serum levels of IL-17F, IL-17A, IL-17A/F were detected in micetreated with 356A11 and 368D04 (FIGS. 22A-C). Also observed was a trendof decreasing IL-6 serum levels in mice treated with 356A11 and 368D04(FIG. 22D). IFNγ and TNFα were below the detection limit in serum frommice treated with control, 356A11, or 368D04 antibodies. Similar resultswere also observed in 356A 11 treated mice with coadministration of LPSand IL-12 at day one following adoptive transfer. (FIGS. 29A-D). In aseparate study, significantly lower serum levels of IL-17A and IL-6 werealso observed in mice treated with 356A 11 without (FIGS. 39A-B) andwith (FIGS. 39C-D) coadministration of LPS and IL-12 at day onefollowing adoptive transfer.

Lymph Node Cell Isolation and Stimulation

Psoriatic lesions in this model usually progress from the ear, eye, faceand neck to the rest of the body. Mice treated with therapeutics usuallydevelop mild skin lesions around the eye and ear only. Therefore,cervical lymph nodes that drain the face were isolated from each mouseto obtain the highest number of activated cells. Lymph node (LN) cellswere recovered by mechanical homogenization. LN cells were pooled fromabout 9 to 10 mice per group and resuspended at 1×10⁶/ml in completeRPMI 1640 medium supplemented with 10% FBS (HyClone), 5×10⁻⁵ M 2-ME(Sigma), 2 mM glutamine (Life Technologies, Gaithersburg, Md.), 10 U/mlpenicillin, 100 μg streptomycin (Life Technologies), and 15 mM HEPES. Atotal of 200 μg/well of this suspension was then placed in a 96-wellplate and stimulated with anti-CD3 plus anti-CD28 (1 μg/ml each) for 48hours.

Intracellular Cytokine Staining

To examine the effects of IL-22 neutralization on the effector T cellpopulation, intracellular cytokine staining was performed on cervicallymph node cells. Cells were stimulated with 50 ng/ml PMA (Sigma), 1μg/ml ionomycin (Sigma), and GolgiPlug (Pharmingen) for 12 hours. Cellswere first stained for surface antigen (CD4) and then treated withCytofix/Cytoperm (Pharmingen) according to manufacturer's directions.Intracellular cytokine staining was performed using antibodies to IFNγ,IL-22, IL-17A, IL-17F, TNFα, and relevant IgG isotype controls.Anti-IL-22 (Ab-02) was labeled with Alexa 647 (Molecular Probes) andanti-IL-17F (RK015-01) was labeled with FITC (Pierce Biotechnologies)according to manufacturer's directions.

Cells were analysed on a gated CD4+ population. FACS analysis resultsshow that in mice treated with 356A11 and 368D04 (withoutcoadministration of LPS and IL-12), there were lower percentages of CD4+T cells producing IL-22, IL-17A, and both IL-22 and IL-17F, but higherpercentages of CD4+ T cells producing IFNγ when compared to the isotypecontrol treated mice (FIG. 23). Similar results were also observed in356A11 treated mice with coadministration of LPS and IL-12 at day onefollowing adoptive transfer. (FIG. 31).

Quantitation of Cytokine Transcripts.

RNA was isolated from the mouse ears or LN cells (after stimulation)using the Qiagen Rneasy kit (Qiagen). Quantitative PCR for cytokinetranscripts was performed using prequalified primers and probes (AppliedBiosystems). The ΔΔCt method was used to normalize transcript to GAPDHand to calculate fold induction relative to control mice.

The quantitative PCR results using RNA from mouse ears show that micetreated with 356A11 and 368D04 had lower IL-22, IL-17F, IL-17A, and IL-6gene expression but enhanced IFNγ expression when compared to micetreated with the control antibody (FIGS. 24A-E). Similar results werealso observed in 356A11 treated mice with coadministration of LPS andIL-12 at day one following adoptive transfer. (FIGS. 30A-E). Theseresults were replicated in a separate study with 356A11 (withoutcoadministration of LPS and IL-12), where it was also observed that356A11 treated mice had lower IL-1 family 6 protein (IL-1F6) geneexpression and unchanged IL-22 binding protein and IL-22 receptorsubunit (IL-22R1) gene expression, as compared to mice treated with thecontrol antibody. (FIGS. 36A-H).

The quantitative PCR results using RNA from lymph node cells, stimulatedwith anti-CD3 and anti-CD28 for 48 hrs, as described above, show that356A11 and 368D04 suppressed IL-22, IL-6, IL-17A, and IL-17F geneexpression ex vivo but affected no significant changes in IFNγ geneexpression. (FIGS. 26A-E). Similar results were observed in 356A11treated mice with coadministration of LPS and IL-12 at day one followingadoptive transfer. (FIGS. 33A-E).

Supernatants from the lymph node cells stimulated with anti-CD3 andanti-CD28, as described above, were collected for cytokine analysisusing ELISA kits (R&D system) as discussed previously. Cytokine ELISAresults mirrored the LN gene expression data, showing that 356A11 and368D04 suppressed IL-22, IL-6, IL-17A, and IL-17F secretion ex vivo butproduced no significant changes in IFNγ secretion from stimulated cells.(FIGS. 25A-F). Similar results were observed in 356A11 treated mice withcoadministration of LPS and IL-12 at day one following adoptivetransfer. (FIGS. 32A-E).

Histopathologic Analysis

Necropsies were performed on mouse tissue upon in vivo studytermination. Tissue samples from ear, trunk skin were collected andfixed in 10% formalin solution for section preparation and analysis. Torecord disease severity, semiquantitive histological scores from 0 to 5were assigned based on the severity of inflammation. Histologicalevaluation was performed in a blinded fashion by a board certifiedpathologist. 0=within normal limit; 1=minimal; 2=mild, 3=moderate,4=marked, and 5=severe.

Immunohistochemistry Analysis

Tissue samples were collected and embedded in Tissue Tek OCT (Miles,Elkhurt, Ind.) compound and frozen with dry ice for cryostat-cutsections. Tissue sections (5 μm) were fixed in 100% acetone and stainedwith anti-CD4, anti-CD11b and anti-neutrophil antibody (PharMingen).Tissues were evaluated as negative=0, mild=1, moderate=2, and severe=3based on visual fluorescent microscopy detection.

Histology findings demonstrate that mice treated with 356A11 and 368D04experienced lesser keratinocyte proliferation, rete pegs, andinflammatory cell infiltrates in the skin when compared to the isotypecontrol treatment. Additionally, immunohistochemistry results showedthat 356A11 decreased the number of CD4+, CD11b+ (macrophages) andneutrophils in the epidermal, dermal, and sub cutis layers. Thehistological results mirrored that of the clinical findings and supportusing IL-22 antagonists, such as the antibodies of this invention, astherapeutic agents for the treatment of psoriasis and otherpsorasis-like skin diseases.

Overall results demonstrate that antibodies of this invention, such as087B03, 368D04, 354A08 or 356A11, are efficacious in this murine modelof psoriasis and indicates that treatment with anti-IL-22 antibodies,including 087B03, 368D04, 354A08 or 356A11, provides an efficaciousstrategy for therapeutic intervention in human psoriasis.

Example 16 Treatment of Patients

Patients with an autoimmune disorder, respiratory disorder, inflammatorycondition of the skin, cardiovascular system, nervous system, kidneys,liver and pancreas or transplant patients are among the type of patientsthat may be treated with the antibodies of the invention. Exemplarytreatment regimens and expected outcomes are provided below. Dosages andfrequencies of administration other than those in Table 14 may also beused. The skilled artisan can adjust treatment regimens as necessarybased on route of administration or other known variables, such as theage, weight, condition, sex, severity of medical condition, etc. of thepatient to be treated.

TABLE 14 Treatment Regimens Dosage Expected Disorder Treated with RangeFrequency Outcome Multiple 087B03, 250 μg/kg weekly, improvement orSclerosis 368D04, to 2 mg/kg biweekly, stabilization of 356A11, or ormonthly condition 354A08 Rheumatoid 087B03, 250 μg/kg weekly,improvement or Arthritis 368D04 to 2 mg/kg biweekly, stabilization of356A11, or or monthly condition 354A08 Psoriasis 087B03, 250 μg/kgweekly, improvement or 368D04, to 2 mg/kg biweekly, stabilization of356A11, or or monthly condition 354A08 IBD 087B03, 250 μg/kg monthly,improvement or 368D04, to 2 mg/kg biweekly, stabilization of 356A11, oror monthly condition 354A08 Alzheimer's 087B03, 250 μg/kg monthly,improvement or Disease 368D04, to 2 mg/kg biweekly, stabilization of356A11, or or monthly condition 354A08

The specification is most thoroughly understood in light of theteachings of the references cited within the specification. Theembodiments within the specification provide an illustration ofembodiments of the invention and should not be construed to limit thescope of the invention. The skilled artisan readily recognizes that manyother embodiments are encompassed by the invention. All publications andpatents cited in this disclosure are incorporated by reference in theirentirety. To the extent the material incorporated by referencecontradicts or is inconsistent with this specification, thespecification will supercede any such material. The citation of anyreferences herein is not an admission that such references are prior artto the present invention.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in thespecification, including claims, are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless otherwiseindicated to the contrary, the numerical parameters are approximationsand may vary depending upon the desired properties sought to be obtainedby the present invention. At the very least, and not as an attempt tolimit the application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should be construed in light of thenumber of significant digits and ordinary rounding approaches.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of treating an interleukin-22 (IL-22)-associated disorder,in a subject, wherein the IL-22-associated disorder is arthritis orpsoriasis, said method comprising, administering to the subject anantibody, or antigen-binding fragment thereof, that specifically bindsto human IL-22, in an amount sufficient to inhibit or reduce immune cellactivity in the subject, thereby treating the disorder, wherein theantibody, or antigen-binding fragment thereof, comprises a V_(H) domaincomprising three complementary determining regions (CDRs) comprising theamino acid sequences of SEQ ID NO:602, 603, and 604 and a V_(L) domaincomprising three CDRs comprising the amino acid sequences of SEQ ID NO:605, 606, and
 607. 2. The method of claim 1, wherein the VL domaincomprises the amino acid sequence of SEQ ID NO:600.
 3. The method ofclaim 2, wherein the V_(H) domain comprises the amino acid sequence ofSEQ ID NO:599.
 4. The method of claim 1, wherein the V_(H) domaincomprises the amino acid sequence of SEQ ID NO:599 and the V_(L) domaincomprises the sequence of SEQ ID NO:600.
 5. The method of claim 1,wherein the antibody, or antigen-binding fragment thereof, has anassociation constant for human IL-22 of at least 10¹⁰ M⁻¹.
 6. The methodof claim 5, wherein the association constant for human IL-22 is at least10¹¹ M⁻¹.
 7. The method of claim 1, wherein the antibody, orantigen-binding fragment thereof, blocks IL-22 mediated BAF3 cellproliferation with an IC₅₀ of 150 pM or less, wherein the BAF3 cellscomprise a human IL-22 receptor.
 8. The method of claim 7, wherein theantibody, or antigen-binding fragment thereof, blocks IL-22 mediatedBAF3 cell proliferation with an IC₅₀ of 100 pM or less.
 9. The method ofclaim 1, wherein the antibody, or antigen-binding fragment thereof,blocks IL-22 mediated GROa secretion from HT29 cells with an IC₅₀ of 1nM or less.
 10. The method of claim 9, wherein the antibody, orantigen-binding fragment thereof, blocks IL-22 mediated GROa secretionfrom HT29 cells with an IC₅₀ of 150 pM or less.
 11. The method of claim1, wherein the subject is a mammal.
 12. The method of claim 1, whereinthe subject is a human.
 13. The method of claim 12, wherein the antibodyis administered in a range chosen from 1 μg/kg to 20 mg/kg, 1 μg/kg to10 mg/kg, 1 μg/kg to 1 mg/kg, 10 μg/kg to 1 mg/kg, 10 μg/kg to 100μg/kg, 100 μg to 1 mg/kg, 250 μg/kg to 2 mg/kg, 250 μg/kg to 1 mg/kg,500 μg/kg to 2 mg/kg, 1 mg/kg to 5 mg/kg, 5 mg/kg to 10 mg/kg, 10 mg/kgto 20 mg/kg, 10 mg/kg to 25 mg/kg, and 20 mg/kg to 30 mg/kg.
 14. Themethod of claim 1, wherein the IL-22-associated disorder is arthritis.15. The method of claim 1, wherein the IL-22-associated disorder ispsoriasis.
 16. A method of treating arthritis or psoriasis in a subject,said method comprising, administering to the subject an antibody, orantigen-binding fragment thereof, that specifically binds to humanIL-22, in an amount sufficient to inhibit or reduce immune cell activityin the subject, thereby treating the arthritis or psoriasis, wherein theantibody, or antigen-binding fragment thereof, comprises a V_(H) domaincomprising an amino acid sequence that is at least 85% identical to SEQID NO:599 and a V_(L) domain comprising an amino acid sequence that isat least 85% identical to SEQ ID NO:600.
 17. The method of claim 16,wherein the V_(H) domain comprises an amino acid sequence that is atleast 90% identical to SEQ ID NO:599 and the V_(L) domain comprises anamino acid sequence that is at least 90% identical to SEQ ID NO:600. 18.The method of claim 16, wherein the V_(H) domain comprises an amino acidsequence that is at least 95% identical to SEQ ID NO:599 and the V_(L)domain comprises an amino acid sequence that is at least 95% identicalto SEQ ID NO:600.
 19. The method of claim 16, wherein the V_(H) domaincomprises an amino acid sequence that is at least 98% identical to SEQID NO:599 and the V_(L) domain comprises an amino acid sequence that isat least 98% identical to SEQ ID NO:600.
 20. The method of claim 16,wherein the V_(H) domain differs by no more than 5 amino acids from theamino acid sequence of SEQ ID NO:599 and wherein the V_(L) domaindiffers by no more than 5 amino acids from the amino acid sequence ofSEQ ID NO:600.
 21. The method of claim 16, wherein the method comprisestreating arthritis.
 22. The method of claim 16, wherein the methodcomprises treating psoriasis.
 23. The method of claim 16, wherein theV_(H) domain comprises the amino acid sequence of SEQ ID NO:599 and theV_(L) domain comprises the amino acid sequence of SEQ ID NO:600.
 24. Amethod of treating arthritis or psoriasis in a subject, said methodcomprising, administering to the subject an antibody, or antigen-bindingfragment thereof, that specifically binds to human IL-22, in an amountsufficient to inhibit or reduce immune cell activity in the subject,thereby treating the arthritis or psoriasis, wherein the antibody, orantigen-binding fragment thereof, comprises a V_(H) domain comprising anamino acid sequence that is at least 85% identical to SEQ ID NO:599. 25.The method of claim 24, wherein the V_(H) domain comprises an amino acidsequence that is at least 95% identical to SEQ ID NO:599.
 26. The methodof claim 24, wherein the V_(H) domain comprises an amino acid sequencethat is at least 98% identical to SEQ ID NO:599.
 27. The method of claim24, wherein the V_(H) domain comprises the amino acid sequence of SEQ IDNO:599.
 28. A method of treating arthritis or psoriasis in a subject,said method comprising, administering to the subject an antibody, orantigen-binding fragment thereof, that specifically binds to humanIL-22, in an amount sufficient to inhibit or reduce immune cell activityin the subject, thereby treating the arthritis or psoriasis, wherein theantibody, or antigen-binding fragment thereof, comprises a V_(L) domaincomprising an amino acid sequence that is at least 85% identical to SEQID NO:600.
 29. The method of claim 28, wherein the V_(L) domaincomprises an amino acid sequence that is at least 95% identical to SEQID NO:600.
 30. The method of claim 28, wherein the V_(L) domaincomprises an amino acid sequence that is at least 98% identical to SEQID NO:600.
 31. The method of claim 28, wherein the V_(L) domaincomprises the amino acid sequence of SEQ ID NO:600.